5-5 fused rings as C5a inhibitors

ABSTRACT

The present disclosure provides, inter alia, Compounds of Formula (I) 
                         
or pharmaceutically acceptable salts thereof that are modulators of the C5a receptor. Also provided are pharmaceutical compositions and methods of use including the treatment of diseases or disorders involving pathologic activation from C5a and non-pharmaceutical applications.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is an application claiming benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 62/513,025 filed May 31,2017, which is herein incorporated by reference in its entirety.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not Applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not Applicable

BACKGROUND OF THE INVENTION

The complement system plays a central role in the clearance of immunecomplexes and in immune responses to infectious agents, foreignantigens, virus infected cells and tumor cells. Inappropriate orexcessive activation of the complement system can lead to harmful, andeven potentially life-threatening consequences due to severeinflammation and resulting tissue destruction. These consequences areclinically manifested in various disorders including septic shock;myocardial, as well as, intestinal ischemia/reperfusion injury; graftrejection; organ failure; nephritis; pathological inflammation; andautoimmune diseases.

The complement system is composed of a group of proteins that arenormally present in the serum in an inactive state. Activation of thecomplement system encompasses mainly three distinct pathways, i.e., theclassical, the alternative, and the lectin pathway (V. M. Holers, InClinical Immunology: Principles and Practice, ed. R. R. Rich, MosbyPress; 1996, 363-391): 1) The classical pathway is acalcium/magnesium-dependent cascade, which is normally activated by theformation of antigen-antibody complexes. It can also be activated in anantibody-independent manner by the binding of C-reactive protein,complexed with ligand, and by many pathogens including gram-negativebacteria. 2) The alternative pathway is a magnesium-dependent cascadewhich is activated by deposition and activation of C3 on certainsusceptible surfaces (e.g. cell wall polysaccharides of yeast andbacteria, and certain biopolymer materials). 3) The lectin pathwayinvolves the initial binding of mannose-binding lectin and thesubsequent activation of C2 and C4, which are common to the classicalpathway (Matsushita, M. et al., J. Exp. Med. 176: 1497-1502 (1992);Suankratay, C. et al., J. Immunol. 160: 3006-3013 (1998)).

The activation of the complement pathway generates biologically activefragments of complement proteins, e.g. C3a, C4a and C5a anaphylatoxinsand C5b-9 membrane attack complexes (MAC), all which mediateinflammatory responses by affecting leukocyte chemotaxis; activatingmacrophages, neutrophils, platelets, mast cells and endothelial cells;and increasing vascular permeability, cytolysis and tissue injury.

Complement C5a is one of the most potent proinflammatory mediators ofthe complement system. (The anaphylactic C5a peptide is 100 times morepotent, on a molar basis, in eliciting inflammatory responses than C3a.)C5a is the activated form of C5 (190 kD, molecular weight). C5a ispresent in human serum at approximately 80 μg/ml (Kohler, P. F. et al.,J. Immunol. 99: 1211-1216 (1967)). It is composed of two polypeptidechains, a and 3, with approximate molecular weights of 115 kD and 75 kD,respectively (Tack, B. F. et al., Biochemistry 18: 1490-1497 (1979)).Biosynthesized as a single-chain promolecule, C5 is enzymaticallycleaved into a two-chain structure during processing and secretion.After cleavage, the two chains are held together by at least onedisulphide bond as well as noncovalent interactions (Ooi, Y. M. et al.,J. Immunol. 124: 2494-2498(1980)).

C5 is cleaved into the C5a and C5b fragments during activation of thecomplement pathways. The convertase enzymes responsible for C5activation are multi-subunit complexes of C4b, C2a, and C3b for theclassical pathway and of (C3b)₂, Bb, and P for the alternative pathway(Goldlust, M. B. et al., J. Immunol. 113: 998-1007 (1974); Schreiber, R.D. et al, Proc. Natl. Acad. Sci. 75: 3948-3952 (1978)). C5 is activatedby cleavage at position 74-75 (Arg-Leu) in the α-chain. Afteractivation, the 11.2 kD, 74 amino acid peptide C5a from theamino-terminus portion of the α-chain is released. Both C5a and C3a arepotent stimulators of neutrophils and monocytes (Schindler, R. et al.,Blood 76: 1631-1638 (1990); Haeffner-Cavaillon, N. et al., J. Immunol.138: 794-700 (1987); Cavaillon, J. M. et al., Eur. J. Immunol. 20:253-257 (1990)).

In addition to its anaphylatoxic properties, C5a induces chemotacticmigration of neutrophils (Ward, P. A. et al., J. Immunol. 102: 93-99(1969)), eosinophils (Kay, A. B. et al., Immunol. 24: 969-976 (1973)),basophils (Lett-Brown, M. A. et al., J. Immunol. 117: 246-252 1976)),and monocytes (Snyderman, R. et al., Proc. Soc. Exp. Biol. Med. 138:387-390 1971)). Both C5a and C5b-9 activate endothelial cells to expressadhesion molecules essential for sequestration of activated leukocytes,which mediate tissue inflammation and injury (Foreman, K. E. et al., J.Clin. Invest. 94: 1147-1155 (1994); Foreman, K. E. et al., Inflammation20: 1-9 (1996); Rollins, S. A. et al., Transplantation 69: 1959-1967(2000)). C5a also mediates inflammatory reactions by causing smoothmuscle contraction, increasing vascular permeability, inducing basophiland mast cell degranulation and inducing release of lysosomal proteasesand oxidative free radicals (Gerard, C. et al., Ann. Rev. Immunol. 12:775-808 (1994)). Furthermore, C5a modulates the hepatic acute-phase geneexpression and augments the overall immune response by increasing theproduction of TNF-α, IL-1-(3, IL-6, IL-8, prostaglandins andleukotrienes (Lambris, J. D. et al., In: The Human Complement System inHealth and Disease, Volanakis, J. E. ed., Marcel Dekker, New York, pp.83-118).

The anaphylactic and chemotactic effects of C5a are believed to bemediated through its interaction with the C5a receptor. The human C5areceptor (C5aR) is a 52 kD membrane bound G protein-coupled receptor,and is expressed on neutrophils, monocytes, basophils, eosinophils,hepatocytes, lung smooth muscle and endothelial cells, and renalglomerular tissues (Van-Epps, D. E. et al., J. Immunol. 132: 2862-2867(1984); Haviland, D. L. et al., J. Immunol. 154:1861-1869 (1995);Wetsel, R. A., Immunol. Leff. 44: 183-187 (1995); Buchner, R. R. et al.,J. Immunol. 155: 308-315 (1995); Chenoweth, D. E. et al., Proc. Natl.Acad. Sci. 75: 3943-3947 (1978); Zwirner, J. et al., Mol. Immunol.36:877-884 (1999)). The ligand-binding site of C5aR is complex andconsists of at least two physically separable binding domains. One bindsthe C5a amino terminus (amino acids 1-20) and disulfide-linked core(amino acids 21-61), while the second binds the C5a carboxy-terminal end(amino acids 62-74) (Wetsel, R. A., Curr. Opin. Immunol. 7: 48-53(1995)).

C5a plays important roles in inflammation and tissue injury. Incardiopulmonary bypass and hemodialysis, C5a is formed as a result ofactivation of the alternative complement pathway when human blood makescontact with the artificial surface of the heart-lung machine or kidneydialysis machine (Howard, R. J. et al., Arch. Surg. 123: 1496-1501(1988); Kirklin, J. K. et al., J. Cardiovasc. Surg. 86: 845-857 (1983);Craddock, P. R. et al., N. Engl. J. Med. 296: 769-774 (1977)). C5acauses increased capillary permeability and edema, bronchoconstriction,pulmonary vasoconstriction, leukocyte and platelet activation andinfiltration to tissues, in particular the lung (Czermak, B. J. et al.,J. Leukoc. Biol. 64: 40-48 (1998)). Administration of an anti-C5amonoclonal antibody was shown to reduce cardiopulmonary bypass andcardioplegia-induced coronary endothelial dysfunction (Tofukuji, M. etal., J. Thorac. Cardiovasc. Surg. 116: 1060-1068 (1998)).

C5a is also involved in acute respiratory distress syndrome (ARDS),Chronic Obstructive Pulmonary Disorder (COPD) and multiple organ failure(MOF) (Hack, C. E. et al., Am. J. Med. 1989: 86: 20-26; Hammerschmidt DE et al. Lancet 1980; 1: 947-949; Heideman M. et al. J. Trauma 1984; 4:1038-1043; Marc, M M, et al., Am. J. Respir. Cell and Mol. Biol., 2004:31: 216-219). C5a augments monocyte production of two importantpro-inflammatory cytokines, TNF-α and IL-1. C5a has also been shown toplay an important role in the development of tissue injury, andparticularly pulmonary injury, in animal models of septic shock(Smedegard G et al. Am. J. Pathol. 1989; 135: 489-497; Markus, S., etal., FASEB Journal (2001), 15: 568-570). In sepsis models using rats,pigs and non-human primates, anti-C5a antibodies administered to theanimals before treatment with endotoxin or E. coli resulted in decreasedtissue injury, as well as decreased production of IL-6 (Smedegard, G. etal., Am. J Pathol. 135: 489-497 (1989); Hopken, U. et al., Eur. J.Immunol. 26: 1103-1109 (1996); Stevens, J. H. et al., J. Clin. Invest.77: 1812-1816 (1986)). More importantly, blockade or C5a with anti-C5apolyclonal antibodies has been shown to significantly improve survivalrates in a caecal ligation/puncture model of sepsis in rats (Czermak, B.J. et al., Nat. Med. 5: 788-792 (1999)). This model share many aspectsof the clinical manifestation of sepsis in humans. (Parker, S. J. etal., Br. J. Surg. 88: 22-30 (2001)). In the same sepsis model, anti-C5aantibodies were shown to inhibit apoptosis of thymocytes (Guo, R. F. etal., J. Clin. Invest. 106: 1271-1280 (2000)) and prevent MOF(Huber-Lang, M. et al., J. Immunol. 166: 1193-1199 (2001)). Anti-C5aantibodies were also protective in a cobra venom factor model of lunginjury in rats, and in immune complex-induced lung injury (Mulligan, M.S. et al. J. Clin. Invest. 98: 503-512 (1996)). The importance of C5a inimmune complex-mediated lung injury was later confirmed in mice (Bozic,C. R. et al., Science 26: 1103-1109 (1996)).

C5a is found to be a major mediator in myocardial ischemia-reperfusioninjury. Complement depletion reduced myocardial infarct size in mice(Weisman, H. F. et al., Science 249: 146-151 (1990)), and treatment withanti-C5a antibodies reduced injury in a rat model of hindlimbischemia-reperfusion (Bless, N. M. et al., Am. J. Physiol. 276: L57-L63(1999)). Reperfusion injury during myocardial infarction was alsomarkedly reduced in pigs that were retreated with a monoclonal anti-C5aIgG (Amsterdam, E. A. et al., Am. J. Physiol. 268:H448-H457 (1995)). Arecombinant human C5aR antagonist reduces infarct size in a porcinemodel of surgical revascularization (Riley, R. D. et al., J. Thorac.Cardiovasc. Surg. 120: 350-358 (2000)).

C5a driven neutrophils also contribute to many bullous diseases (e.g.,bullous pemphigoid, pemphigus vulgaris and pemphigus foliaceus). Theseare chronic and recurring inflammatory disorders clinicallycharacterized by sterile blisters that appear in the sub-epidermal spaceof the skin and mucosa. While autoantibodies to keratinocytes located atthe cutaneous basement membranes are believed to underlie the detachmentof epidermal basal keratinocytes from the underlying basement membrane,blisters are also characterized by accumulation of neutrophils in boththe upper dermal layers and within the blister cavities. In experimentalmodels a reduction of neutrophils or absence of complement (total orC5-selective) can inhibit formation of sub-epidermal blisters, even inthe presence of high auto-antibody titers.

Complement levels are elevated in patients with rheumatoid arthritis(Jose, P. J. et al., Ann. Rheum. Dis. 49: 747-752 (1990); Grant, E. P.,et al., J. of Exp. Med., 196(11): 1461-1471, (2002)), lupus nephritis(Bao, L., et al., Eur. J. of Immunol., 35(8), 2496-2506, (2005)) andsystemic lupus erythematosus (SLE) (Porcel, J. M. et al., Clin. Immunol.Immunopathol. 74: 283-288 (1995)). C5a levels correlate with theseverity of the disease state. Collagen-induced arthritis in mice andrats resembles the rheumatoid arthritic disease in human. Mice deficientin the C5a receptor demonstrated a complete protection from arthritisinduced by injection of monoclonal anti-collagen Abs (Banda, N. K., etal., J. of Immunol., 2003, 171: 2109-2115). Therefore, inhibition of C5aand/or C5a receptor (C5aR) could be useful in treating these chronicdiseases.

The complement system is believed to be activated in patients withinflammatory bowel disease (IBD) and is thought to play a role in thedisease pathogenesis. Activated complement products were found at theluminal face of surface epithelial cells, as well as in the muscularismucosa and submucosal blood vessels in IBD patients (Woodruff, T. M., etal., J of Immunol., 2003, 171: 5514-5520).

C5aR expression is upregulated on reactive astrocytes, microglia, andendothelial cells in an inflamed human central nervous system (Gasque,P. et al., Am. J. Pathol. 150: 31-41 (1997)). C5a might be involved inneurodegenerative diseases, such as Alzheimer disease (Mukherjee, P. etal., J. Neuroimmunol. 105: 124-130 (2000); O'Barr, S. et al., J.Neuroimmunol. (2000) 105: 87-94; Farkas, I., et al. J. Immunol. (2003)170:5764-5771), Parkinson's disease, Pick disease and transmissiblespongiform encephalopathies. Activation of neuronal C5aR may induceapoptosis (Farkas I et al. J. Physiol. 1998; 507: 679-687). Therefore,inhibition of C5a and/or C5aR could also be useful in treatingneurodegenerative diseases.

There is some evidence that C5a production worsens inflammationassociated with atopic dermatitis (Neuber, K., et al., Immunology73:83-87, (1991)), and chronic urticaria (Kaplan, A. P., J. AllergyClin. Immunol. 114; 465-474, (2004).

Psoriasis is now known to be a T cell-mediated disease (Gottlieb, E. L.et al., Nat. Med. 1: 442-447 (1995)). However, neutrophils and mastcells may also be involved in the pathogenesis of the disease (Terui, T.et al., Exp. Dermatol. 9: 1-10; 2000); Werfel, T. et al., Arch.Dermatol. Res. 289: 83-86 (1997)). Neutrophil accumulation under thestratum corneum is observed in the highly inflamed areas of psoriaticplaques, and psoriatic lesion (scale) extracts contain highly elevatedlevels of C5a and exhibit potent chemotactic activity towardsneutrophils, an effect that can be inhibited by addition of a C5aantibody. T cells and neutrophils are chemo-attracted by C5a (Nataf, S.et al., J. Immunol. 162: 4018-4023 (1999); Tsuji, R. F. et al., J.Immunol. 165: 1588-1598 (2000); Cavaillon, J. M. et al., Eur. J.Immunol. 20: 253-257 (1990)). Additionally expression of C5aR has beendemonstrated in plasmacytoid dendritic cells (pDC) isolated from lesionsof cutaneous lupus erythematous and these cells were shown to displaychemotactic behavior towards C5a, suggesting that blockade of C5aR onpDC might be efficacious in reducing pDC infiltration into inflamed skinin both SLE and psoriasis. Therefore C5a could be an importanttherapeutic target for treatment of psoriasis.

Immunoglobulin G-containing immune complexes (IC) contribute to thepathophysiology in a number of autoimmune diseases, such as systemiclupus erthyematosus, rheumatoid arthritis, Sjogren's disease,Goodpasture's syndrome, and hypersensitivity pneumonitis (Madaio, M. P.,Semin. Nephrol. 19: 48-56 (1999); Korganow, A. S. et al., Immunity 10:451-459 (1999); Bolten, W. K., Kidney nt. 50: 1754-1760 (1996); Ando, M.et al., Curr. Opin. Pulm. Med. 3: 391-399 (1997)). These diseases arehighly heterogeneous and generally affect one or more of the followingorgans: skin, blood vessels, joints, kidneys, heart, lungs, nervoussystem and liver (including cirrhosis and liver fibrosis). The classicalanimal model for the inflammatory response in these IC diseases is theArthus reaction, which features the infiltration of polymorphonuclearcells, hemorrhage, and plasma exudation (Arthus, M., C. R. Soc. Biol.55: 817-824 (1903)). Recent studies show that C5aR deficient mice areprotected from tissue injury induced by IC (Kohl, J. et al., Mol.Immunol. 36: 893-903 (1999); Baumann, U. et al., J. Immunol. 164:1065-1070 (2000)). The results are consistent with the observation thata small peptidic anti-C5aR antagonist inhibits the inflammatory responsecaused by IC deposition (Strachan, A. J. et al., J. Immunol. 164:6560-6565 (2000)). Together with its receptor, C5a plays an importantrole in the pathogenesis of IC diseases. Inhibitors of C5a and C5aRcould be useful to treat these diseases.

Description of Related Art

Non-peptide based C5a receptor antagonist have been reported as beingeffective for treating endotoxic shock in rats (Stracham, A. J., et al.,J. of Immunol. (2000), 164(12): 6560-6565); and for treating IBD in arat model (Woodruff, T. M., et al., J of Immunol., 2003, 171:5514-5520). Non-peptide based C5a receptor modulators also have beendescribed in the patent literature by Neurogen Corporation, (e.g.,WO2004/043925, WO2004/018460, WO2005/007087, WO03/082826, WO03/08828,WO02/49993, WO03/084524); Dompe S. P. A. (WO02/029187); The Universityof Queenland (WO2004/100975); and ChemoCentryx (WO2010/075257).

There is considerable experimental evidence in the literature thatimplicates increased levels of C5a with a number of diseases anddisorders, in particular in autoimmune and inflammatory diseases anddisorders. Thus, there remains a need in the art for new small organicmolecule modulators, e.g., agonists, preferably antagonists, partialagonists, of the C5a receptor (C5aR) that are useful for inhibitingpathogenic events, e.g., chemotaxis, associated with increased levelsanaphylatoxin activity. The present invention fulfills this and otherneeds.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provide compounds of Formula (I):

or a pharmaceutically acceptable salt thereof, wherein,

-   -   ring vertex A¹ is selected from the group consisting of N, CH,        C(O) and C(R⁴);    -   ring vertex A² is selected from the group consisting of N, CH,        and C(R⁴);    -   each of ring vertices A³, A⁴, A⁵ and A⁶ is independently        selected from the group consisting of CH and C(R⁴);    -   each of the dashed bonds independently indicate a single or        double bond;    -   R¹ is selected from the group consisting of —C₁₋₈        alkylene-heteroaryl, —C₁₋₈ alkylene-C₆₋₁₀ aryl, C₁₋₈ alkyl, C₁₋₈        haloalkyl, —C(O)—C₁₋₈ alkyl, —C(O)—C₆₋₁₀ aryl, —C(O)-heteroaryl,        —C(O)—C₃₋₆ cycloalkyl, —C(O)-heterocycloalkyl,        —C(O)NR^(1a)R^(1b), —SO₂—C₆₋₁₀ aryl, —SO₂-heteroaryl, —C(O)—C₁₋₈        alkylene-O-heteroaryl, —C(O)—C₁₋₈ alkylene-O—C₆₋₁₀ aryl,        —C(O)—C₁₋₈ alkylene-O— heterocycloalkyl, —C(O)—C₁₋₈ alkylene-O—        C₃₋₆ cycloalkyl, —C(O)—C₁₋₈ alkylene-heteroaryl, —C(O)—C₁₋₈        alkylene-C₆₋₁₀ aryl, —C(O)—C₁₋₈ alkylene-heterocycloalkyl,        —C(O)—C₁₋₈ alkylene-C₃₋₆ cycloalkyl and —CO₂R^(1a); the        heterocycloalkyl is a 4 to 8 membered ring having from 1 to 3        heteroatoms as ring vertices selected from N, O and S; and the        heteroaryl group is a 5 to 10 membered aromatic ring having from        1 to 3 heteroatoms as ring vertices selected from N, O and S;    -   wherein R^(1a) and R^(1b) are each independently selected from        the group consisting of hydrogen, C₁₋₈ alkyl, and C₁₋₈        haloalkyl;    -   wherein R¹ is optionally substituted with 1 to 5 R⁵        substituents;    -   R^(2a) and R^(2e) are each independently selected from the group        consisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, —O—C₁₋₆        haloalkyl, —S—C₁₋₆ alkyl, —C₁₋₆ alkyl-O—C₁₋₆ alkyl, —C₁₋₆        alkyl-S—C₁₋₆ alkyl, CN, and halogen;    -   R^(2b), R^(2c), and R^(2d) are each independently selected from        the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆        haloalkyl, —O—C₁₋₆ haloalkyl, —S—C₁₋₆ alkyl, —C₁₋₆ alkyl-O—C₁₋₆        alkyl, —C₁₋₆ alkyl-S—C₁₋₆ alkyl, cyano, and halogen;    -   each R³ is independently selected from the group consisting of        C₁₋₆ alkyl, C₁₋₆ haloalkyl, halogen and hydroxyl, and optionally        two R³ groups on the same carbon atom are combined to form oxo        (═O) or to form a three to five membered cycloalkyl ring;    -   each R⁴ is independently selected from the group consisting of        C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ hydroxyalkyl, C₁₋₆ haloalkyl, C₁₋₆        haloalkoxy, —O—C₁₋₆ haloalkyl, halogen, cyano, hydroxyl, —S—C₁₋₆        alkyl, —C₁₋₆ alkyl-O—C₁₋₆ alkyl, —C₁₋₆ alkyl-S—C₁₋₆ alkyl,        —NR^(4a)R^(4b), —CONR^(4a)R^(4b), —CO₂R^(4a), —COR^(4a),        —OC(O)NR^(4a)R^(4b), —NR^(4a)C(O)R^(4b), —NR^(4a)C(O)₂R^(4b),        and —NR^(4a)—C(O)NR^(4a)R^(4b);    -   each R^(4a) and R^(4b) is independently selected from the group        consisting of hydrogen, C₁₋₄ alkyl, and C₁₋₄ haloalkyl;    -   each R⁵ is independently selected from the group consisting of        C₁₋₈ alkyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₁₋₈ haloalkoxy, C₁₋₈        hydroxyalkyl, —C₁₋₈ alkylene-heterocycloalkyl, —C₁₋₈        alkylene-C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl, heterocycloalkyl,        halogen, OH, C₂₋₈ alkenyl, C₂₋₈ alkynyl, CN, C(O)R^(5a),        —NR^(5b)C(O)R^(5a), —CONR^(5a)R^(5b), —NR^(5a)R^(5b), —C₁₋₈        alkylene-NR^(5a)R^(5b), —S—C₁₋₆ alkyl, —C₁₋₆ alkylene-O—C₁₋₆        alkyl, —C₁₋₆ alkylene-S—C₁₋₆ alkyl, —OC(O)NR^(5a)R^(5b),        —NR^(5a)C(O)₂R^(5b), —NR^(5a)—C(O)NR^(5a)R^(5b), and CO₂R^(5a);        wherein the heterocycloalkyl group is a 4 to 8 membered ring        having from 1 to 3 heteroatoms as ring vertices selected from N,        O, and S;    -   wherein each R^(5a) and R^(5b) is independently selected from        the group consisting of hydrogen, C₁₋₈ alkyl, and C₁₋₈        haloalkyl, or when R^(5a) and R^(5b) are attached to the same        nitrogen atom they are combined with the nitrogen atom to form a        5 or 6-membered ring having from 0 to 1 additional heteroatoms        as ring vertices selected from N, O, or S; and    -   the subscript n is 0, 1, 2 or 3.

In addition to the compounds provided herein, the present inventionfurther provides pharmaceutical compositions containing one or more ofthese compounds, as well as methods for the use of these compounds intherapeutic methods, primarily to treat diseases associated C5asignaling activity.

In yet another aspect, the present invention provides methods ofdiagnosing disease in an individual. In these methods, the compoundsprovided herein are administered in labeled form to a subject, followedby diagnostic imaging to determine the presence or absence of C5aRand/or the localization of cells expressing a C5aR receptor. In arelated aspect, a method of diagnosing disease is carried out bycontacting a tissue or blood sample with a labeled compound as providedherein and determining the presence, absence, amount, or localization ofC5aR in the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

Not Applicable.

DETAILED DESCRIPTION OF THE INVENTION I. Abbreviation and Definitions

The term “alkyl”, by itself or as part of another substituent, means,unless otherwise stated, a straight or branched chain hydrocarbonradical, having the number of carbon atoms designated (i.e. C₁₋₈ meansone to eight carbons). Examples of alkyl groups include methyl, ethyl,n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. The term “alkenyl” refers toan unsaturated alkyl group having one or more double bonds. Similarly,the term “alkynyl” refers to an unsaturated alkyl group having one ormore triple bonds. Examples of such unsaturated alkyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), isobutenyl,2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl,3-butynyl, and the higher homologs and isomers. The term “cycloalkyl”refers to hydrocarbon rings having the indicated number of ring atoms(e.g., C₃₋₆cycloalkyl) and being fully saturated or having no more thanone double bond between ring vertices. “Cycloalkyl” is also meant torefer to bicyclic and polycyclic hydrocarbon rings such as, for example,bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane, etc. The term“heterocycloalkyl” refers to a cycloalkyl group that contain from one tofive heteroatoms selected from N, O, and S, wherein the nitrogen andsulfur atoms are optionally oxidized, and the nitrogen atom(s) areoptionally quaternized. The heterocycloalkyl may be a monocyclic, abicyclic or a polycyclic ring system. Non limiting examples ofheterocycloalkyl groups include pyrrolidine, imidazolidine,pyrazolidine, butyrolactam, valerolactam, imidazolidinone, hydantoin,dioxolane, phthalimide, piperidine, 1,4-dioxane, morpholine,thiomorpholine, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide,piperazine, pyran, pyridone, 3-pyrroline, thiopyran, pyrone,tetrahydrofuran, tetrhydrothiophene, quinuclidine, and the like. Aheterocycloalkyl group can be attached to the remainder of the moleculethrough a ring carbon or a heteroatom.

The term “alkylene” by itself or as part of another substituent means adivalent radical derived from an alkane, as exemplified by—CH₂CH₂CH₂CH₂—. Typically, an alkyl (or alkylene) group will have from 1to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingfour or fewer carbon atoms. Similarly, “alkenylene” and “alkynylene”refer to the unsaturated forms of “alkylene” having double or triplebonds, respectively.

The term “heteroalkyl,” by itself or in combination with another term,means, unless otherwise stated, a stable straight or branched chain, orcyclic hydrocarbon radical, or combinations thereof, consisting of thestated number of carbon atoms and from one to three heteroatoms selectedfrom the group consisting of O, N, Si and S, and wherein the nitrogenand sulfur atoms may optionally be oxidized and the nitrogen heteroatommay optionally be quaternized. The heteroatom(s) O, N and S may beplaced at any interior position of the heteroalkyl group. The heteroatomSi may be placed at any position of the heteroalkyl group, including theposition at which the alkyl group is attached to the remainder of themolecule. Examples include —CH₂—CH₂—O—CH₃, —CH₂—CH₂—NH—CH₃,—CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂, —S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Similarly, the terms“heteroalkenyl” and “heteroalkynyl” by itself or in combination withanother term, means, unless otherwise stated, an alkenyl group oralkynyl group, respectively, that contains the stated number of carbonsand having from one to three heteroatoms selected from the groupconsisting of O, N, Si and S, and wherein the nitrogen and sulfur atomsmay optionally be oxidized and the nitrogen heteroatom may optionally bequaternized. The heteroatom(s) O, N and S may be placed at any interiorposition of the heteroalkyl group.

The term “heteroalkylene” by itself or as part of another substituentmeans a divalent radical, saturated or unsaturated or polyunsaturated,derived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—, —O—CH₂—CH═CH—, —CH₂—CH═C(H)CH₂—O—CH₂— and—S—CH₂—C≡C—. For heteroalkylene groups, heteroatoms can also occupyeither or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy,alkyleneamino, alkylenediamino, and the like).

The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) areused in their conventional sense, and refer to those alkyl groupsattached to the remainder of the molecule via an oxygen atom, an aminogroup, or a sulfur atom, respectively. Additionally, for dialkylaminogroups, the alkyl portions can be the same or different and can also becombined to form a 3-7 membered ring with the nitrogen atom to whicheach is attached. Accordingly, a group represented as —NR^(a)R^(b) ismeant to include piperidinyl, pyrrolidinyl, morpholinyl, azetidinyl andthe like.

The term “hydroxyalkyl” is used in its conventional sense, and refers tobranched or straight chain alkyl group substituted with at least onehydroxyl group. The hydroxyl group may be at any position in the alkylgroup. For example, the term “C₁₋₄hydroxylalkyl” is meant to includehydroxymethyl, hydroxyethyl, hydroxypropyl, hydroxyisopropyl, and thelike.

The terms “halo” or “halogen,” by themselves or as part of anothersubstituent, mean, unless otherwise stated, a fluorine, chlorine,bromine, or iodine atom. Additionally, terms such as “haloalkyl,” aremeant to include monohaloalkyl and polyhaloalkyl. For example, the term“C₁₋₄ haloalkyl” is mean to include trifluoromethyl,2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like.

The term “aryl” means, unless otherwise stated, a polyunsaturated,typically aromatic, hydrocarbon group which can be a single ring ormultiple rings (up to three rings) which are fused together or linkedcovalently. The term “heteroaryl” refers to aryl groups (or rings) thatcontain from one to five heteroatoms selected from N, O, and S, whereinthe nitrogen and sulfur atoms are optionally oxidized, and the nitrogenatom(s) are optionally quaternized. A heteroaryl group can be attachedto the remainder of the molecule through a heteroatom. Non-limitingexamples of aryl groups include phenyl, naphthyl and biphenyl, whilenon-limiting examples of heteroaryl groups include pyridyl, pyridazinyl,pyrazinyl, pyrimindinyl, triazinyl, quinolinyl, quinoxalinyl,quinazolinyl, cinnolinyl, phthalaziniyl, benzotriazinyl, purinyl,benzimidazolyl, benzopyrazolyl, benzooxazolyl, benzotriazolyl,benzisoxazolyl, isobenzofuryl, isoindolyl, indolizinyl, benzotriazinyl,thienopyridinyl, thienopyrimidinyl, pyrazolopyrimidinyl, pyrrolopyridyl,imidazopyridines, benzothiaxolyl, benzofuranyl, benzothienyl, indolyl,quinolyl, isoquinolyl, isothiazolyl, pyrazolyl, indazolyl, pteridinyl,imidazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiadiazolyl,pyrrolyl, thiazolyl, furyl, thienyl and the like. Substituents for eachof the above noted aryl and heteroaryl ring systems are selected fromthe group of acceptable substituents described below.

The term “pharmaceutically acceptable salts” is meant to include saltsof the active compounds which are prepared with relatively nontoxicacids or bases, depending on the particular substituents found on thecompounds described herein. When compounds of the present inventioncontain relatively acidic functionalities, base addition salts can beobtained by contacting the neutral form of such compounds with asufficient amount of the desired base, either neat or in a suitableinert solvent. Examples of salts derived frompharmaceutically-acceptable inorganic bases include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including substituted amines, cyclicamines, naturally-occurring amines and the like, such as arginine,betaine, caffeine, choline, N,N′-dibenzylethylenediamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethylmorpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperadine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like. When compounds of the presentinvention contain relatively basic functionalities, acid addition saltscan be obtained by contacting the neutral form of such compounds with asufficient amount of the desired acid, either neat or in a suitableinert solvent. Examples of pharmaceutically acceptable acid additionsalts include those derived from inorganic acids like hydrochloric,hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric,monohydrogenphosphoric, dihydrogenphosphoric, sulfuric,monohydrogensulfuric, hydriodic, or phosphorous acids and the like, aswell as the salts derived from relatively nontoxic organic acids likeacetic, propionic, isobutyric, malonic, benzoic, succinic, suberic,fumaric, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric,tartaric, methanesulfonic, and the like. Also included are salts ofamino acids such as arginate and the like, and salts of organic acidslike glucuronic or galactunoric acids and the like (see, for example,Berge, S. M., et al, “Pharmaceutical Salts”, Journal of PharmaceuticalScience, 1977, 66, 1-19). Certain specific compounds of the presentinvention contain both basic and acidic functionalities that allow thecompounds to be converted into either base or acid addition salts.

The neutral forms of the compounds may be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound differs from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

In addition to salt forms, the present invention provides compoundswhich are in a prodrug form. Prodrugs of the compounds described hereinare those compounds that readily undergo chemical changes underphysiological conditions to provide the compounds of the presentinvention. Additionally, prodrugs can be converted to the compounds ofthe present invention by chemical or biochemical methods in an ex vivoenvironment. For example, prodrugs can be slowly converted to thecompounds of the present invention when placed in a transdermal patchreservoir with a suitable enzyme or chemical reagent.

Certain compounds of the present invention can exist in unsolvated formsas well as solvated forms, including hydrated forms. In general, thesolvated forms are equivalent to unsolvated forms and are intended to beencompassed within the scope of the present invention. Certain compoundsof the present invention may exist in multiple crystalline or amorphousforms. In general, all physical forms are equivalent for the usescontemplated by the present invention and are intended to be within thescope of the present invention.

Certain compounds of the present invention possess asymmetric carbonatoms (optical centers) or double bonds; the racemates, diastereomers,geometric isomers, regioisomers and individual isomers (e.g., separateenantiomers) are all intended to be encompassed within the scope of thepresent invention. The compounds of the present invention may alsocontain unnatural proportions of atomic isotopes at one or more of theatoms that constitute such compounds. For example, the compounds may beradiolabeled with radioactive isotopes, such as for example tritium(³H), iodine-125 (¹²⁵I) or carbon-14 (¹⁴C). All isotopic variations ofthe compounds of the present invention, whether radioactive or not, areintended to be encompassed within the scope of the present invention.

As used herein, a wavy line, “

”, that intersects a single, double or triple bond in any chemicalstructure depicted herein, represent the point attachment of the single,double, or triple bond to the remainder of the molecule.

II. Description of the Embodiments A. Compounds

In one aspect, the present invention provides compounds of Formula I:

or a pharmaceutically acceptable salt thereof, wherein,

-   -   ring vertex A¹ is selected from the group consisting of N, CH,        C(O) and C(R⁴);    -   ring vertex A² is selected from the group consisting of N, CH,        and C(R⁴);    -   each of ring vertices A³, A⁴, A⁵ and A⁶ is independently        selected from the group consisting of CH and C(R⁴);    -   each of the dashed bonds independently indicate a single or        double bond;    -   R¹ is selected from the group consisting of —C₁₋₈        alkylene-heteroaryl, —C₁₋₈ alkylene-C₆₋₁₀ aryl, C₁₋₈ alkyl, C₁₋₈        haloalkyl, —C(O)—C₁₋₈ alkyl, —C(O)—C₆₋₁₀ aryl, —C(O)-heteroaryl,        —C(O)—C₃₋₆ cycloalkyl, —C(O)-heterocycloalkyl,        —C(O)NR^(1a)R^(1b), —SO₂—C₆₋₁₀ aryl, —SO₂-heteroaryl, —C(O)—C₁₋₈        alkylene-O-heteroaryl, —C(O)—C₁₋₈ alkylene-O—C₆₋₁₀ aryl,        —C(O)—C₁₋₈ alkylene-O— heterocycloalkyl, —C(O)—C₁₋₈ alkylene-O—        C₃₋₆ cycloalkyl, —C(O)—C₁₋₈ alkylene-heteroaryl, —C(O)—C₁₋₈        alkylene-C₆₋₁₀ aryl, —C(O)—C₁₋₈ alkylene-heterocycloalkyl,        —C(O)—C₁₋₈ alkylene-C₃₆ cycloalkyl and —CO₂R^(1a); the        heterocycloalkyl is a 4 to 8 membered ring having from 1 to 3        heteroatoms as ring vertices selected from N, O and S; and the        heteroaryl group is a 5 to 10 membered aromatic ring having from        1 to 3 heteroatoms as ring vertices selected from N, O and S;    -   wherein R^(1a) and R^(1b) are each independently selected from        the group consisting of hydrogen, C₁₋₈ alkyl, and C₁₋₈        haloalkyl;    -   wherein R¹ is optionally substituted with 1 to 5 R⁵        substituents;    -   R^(2a) and R^(2e) are each independently selected from the group        consisting of C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, —O—C₁₋₆        haloalkyl, —S—C₁₋₆ alkyl, —C₁₋₆ alkyl-O—C₁₋₆ alkyl, —C₁₋₆        alkyl-S—C₁₋₆ alkyl, CN, and halogen;    -   R^(2b), R^(2c), and R^(2d) are each independently selected from        the group consisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆        haloalkyl, —O—C₁₋₆ haloalkyl, —S—C₁₋₆ alkyl, —C₁₋₆ alkyl-O—C₁₋₆        alkyl, —C₁₋₆ alkyl-S—C₁₋₆ alkyl, cyano, and halogen;    -   each R³ is independently selected from the group consisting of        C₁₋₆ alkyl, C₁₋₆ haloalkyl, halogen and hydroxyl, and optionally        two R³ groups on the same carbon atom are combined to form oxo        (═O) or to form a three to five membered cycloalkyl ring;    -   each R⁴ is independently selected from the group consisting of        C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ hydroxyalkyl, C₁₋₆ haloalkyl, C₁₋₆        haloalkoxy, —O—C₁₋₆ haloalkyl, halogen, cyano, hydroxyl, —S—C₁₋₆        alkyl, —C₁₋₆ alkyl-O—C₁₋₆ alkyl, —C₁₋₆ alkyl-S—C₁₋₆ alkyl,        —NR^(4a)R^(4b), —CONR^(4a)R^(4b), —CO₂R^(4a), —COR^(4a),        —OC(O)NR^(4a)R^(4b), —NR^(4a)C(O)R^(4b), —NR^(4a)C(O)₂R^(4b),        and —NR^(4a)—C(O)NR^(4a)R^(4b);    -   each R^(4a) and R^(4b) is independently selected from the group        consisting of hydrogen, C₁₋₄ alkyl, and C₁₋₄ haloalkyl;    -   each R⁵ is independently selected from the group consisting of        C₁₋₈ alkyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₁₋₈ haloalkoxy, C₁₋₈        hydroxyalkyl, —C₁₋₈ alkylene-heterocycloalkyl, —C₁₋₈        alkylene-C₃₋₆ cycloalkyl, C₃₋₆ cycloalkyl, heterocycloalkyl,        halogen, OH, C₂₋₈ alkenyl, C₂₋₈ alkynyl, CN, C(O)R^(5a),        —NR^(5b)C(O)R^(5a), —CONR^(5a)R^(5b), —NR^(5a)R^(5b), —C₁₋₈        alkylene-NR^(5a)R^(5b), —S—C₁₋₆ alkyl, —C₁₋₆ alkylene-O—C₁₋₆        alkyl, —C₁₋₆ alkylene-S—C₁₋₆ alkyl, —OC(O)NR^(5a)R^(5b),        —NR^(5a)C(O)₂R^(5b), —NR^(5a)—C(O)NR^(5a)R^(5b), and CO₂R^(5a);        wherein the heterocycloalkyl group is a 4 to 8 membered ring        having from 1 to 3 heteroatoms as ring vertices selected from N,        O, and S;    -   wherein each R^(5a) and R^(5b) is independently selected from        the group consisting of hydrogen, C₁₋₈ alkyl, and C₁₋₈        haloalkyl, or when R^(5a) and R^(5b) are attached to the same        nitrogen atom they are combined with the nitrogen atom to form a        5 or 6-membered ring having from 0 to 1 additional heteroatoms        as ring vertices selected from N, O, or S; and    -   the subscript n is 0, 1, 2 or 3.

Focusing on the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶ as ringvertices, in some embodiments, the ring portion having A¹, A², A³, A⁴,A⁵, and A⁶ as ring vertices is a bicyclic heteroaryl selected from

-   -   wherein m is 0, 1, 2 or 3; and wherein the R⁴ substituents may        be attached to any suitable carbon ring vertex of the bicyclic        heteroaryl.

In some embodiments, the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶as ring vertices is

-   -   wherein m is 0, 1, 2, or 3; wherein the R⁴ substituents may be        attached to any suitable carbon ring vertex of the bicyclic        heteroaryl.

In some embodiments, each R⁴ is independently C₁₋₄ alkyl, C₁₋₄ alkoxy,C₁₋₆ hydroxyalkyl, halogen, cyano, and —CO₂R^(4a), wherein R^(4a) is asdefined above, and wherein the R⁴ substituents may be attached to anysuitable carbon ring vertex of the bicyclic heteroaryl

A person of skill in the art will recognize that particular carbon atomsof the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶ cannot besubstituted with R⁴. For example, the carbon atom linking the bicyclicheteroaryl moiety (i.e. the ring portion having A¹, A², A³, A⁴, A⁵, andA⁶) to the remainder of the molecule and the carbon atoms that aremembers of both ring systems in the fused bicyclic heteroaryl moiety(i.e. the two carbon atoms that are ring vertices in both the benzeneand five-membered ring system) cannot be substituted with R⁴ because anadditional substituent will exceed the valence of these carbon atoms.

In some embodiments, the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶as ring vertices is selected from the group consisting of:

In some embodiments, the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶as ring vertices is selected from the group consisting of:

Turning to R¹ and optional substituent(s), R⁵, in some embodiments, R¹is —C₁₋₈ alkylene-heteroaryl, —C₁₋₈alkylene-C₆₋₁₀ aryl, C₁₋₈ alkyl, C₁₋₈haloalkyl, —C(O)—C₁₋₈ alkyl, —C(O)—C₆₋₁₀ aryl, —C(O)-heteroaryl,—C(O)—C₃₋₈ cycloalkyl, —C(O)NR^(1a)R^(1b), —SO₂—C₆₋₁₀ aryl, —C(O)—C₁₋₈alkylene-O—C₆₋₁₀ aryl, or —CO₂R^(1a); wherein R^(1a) and R^(1b),heterocycloalkyl, and the heteroaryl are as defined above, and whereinR¹ is optionally substituted with 1 to 5 R⁵ substituents.

In some embodiments, heterocycloalkyl groups of R¹ or R⁵ are from 4 to 6membered rings having from 1 to 3 heteroatoms as ring vertices selectedfrom N, O, and S. In some embodiments, the heteroaryl groups of R¹ or R⁵are 5 to 6 membered aromatic rings having from 1 to 3 heteroatoms asring vertices selected from N, O, and S. In some embodiments, the C₆₋₁₀aryl group of R¹ is phenyl.

In some embodiments, R¹ is —CH₂-phenyl optionally substituted by 1 to 3R⁵.

In some embodiments, each R⁵ is independently of C₁₋₈ alkyl, C₁₋₈alkoxy, C₁₋₈ haloalkyl, C₁₋₈ haloalkoxy, C₁₋₈ hydroxyalkyl, C₃₋₆cycloalkyl, halogen, OH, —NR^(5a)R^(5b), or CO₂R^(5a) wherein eachR^(5a) and R^(5b) is independently selected from the group consisting ofhydrogen, C₁₋₈alkyl, and C₁₋₈ haloalkyl.

In some embodiments, R¹ is —CH₂-phenyl substituted by 1 or 2 R⁵, whereineach R⁵ is independently C₁₋₄ haloalkyl.

In some embodiments, R¹ is —CH₂-phenyl substituted by 1 or 2 CF₃.

In some embodiments, R¹ is selected from the group consisting of

In some embodiments R¹ is

Returning to Formula I and substituents R^(2a), R^(2b), R^(2c), R^(2d),and R^(2e), in some embodiments, R^(2a) and R^(2e) are eachindependently C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₆ alkoxy, —O—C₁₋₆haloalkyl, or halogen. In some embodiments, R^(2b), R^(2c), and R^(2d)are independently H, C₁₋₄ alkyl, C₁₋₄ haloalkyl, or halogen.

In some embodiments, R^(2b), R^(2c), and R^(2d) are each H.

In some embodiments, R^(2a) and R^(2e) are each independently C₁₋₆ alkylor C₁₋₆ haloalkyl.

In some embodiments, R^(2a) and R^(2e) are each independently methyl orethyl. In some embodiments, R^(2a) and R^(2e) are both methyl or areboth ethyl.

In some embodiments, the portion of Formula I represented by

Each R³ of Formula I, in some embodiments, is independently C₁₋₆ alkyl,C₁₋₆ haloalkyl, or halogen. In some embodiments, each R³ isindependently C₁₋₄ alkyl.

In some embodiments, n, the subscript of R³, is 0, 1, or 2. In someembodiments, n is 0. In some embodiments, n is 2.

In some embodiments, the portion of Formula I represented by

In some embodiments, the compound of Formula I is represented by Formula(Ia), (Ib), or (Ic).

In embodiments where the compound of Formula (I) is represented byFormula (Ia), R¹, R³, n, R^(2a), R^(2e), and the ring portion having A¹,A², A³, A⁴, A⁵, and A⁶ as ring vertices are as defined above for Formula(I).

In embodiments where the compound of Formula (I) is represented byFormula (Ib), R¹, R^(2a), R^(2e), and the ring portion having A¹, A²,A³, A⁴, A⁵, and A⁶ as ring vertices are as defined above for Formula(I).

In embodiments where the compound of Formula (I) is represented byFormula (Ic), R¹ and the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶as ring vertices are as defined above for Formula (I).

In some embodiments, the compound of Formula (I) is represented byFormula (Id), (Ie), or (If)

In embodiments where the compound of Formula (I) is represented byFormula (Id), R¹, R⁴, m, R^(2a), and R^(2e) are as defined above forFormula (I).

In embodiments where the compound of Formula (I) is represented byFormula (Ie), R¹, R⁴, and m are as defined above for Formula (I).

In embodiments where the compound of Formula (I) is represented byFormula (If), R⁵, R⁴, and m are as defined above for Formula (I), and pis 0, 1, or 2.

In some embodiments, the compound of Formula (I) is represented byFormula (Ig) or (Ih)

In embodiments where the compound of Formula (I) is represented byFormula (Ig) or (Ih), R⁴ and m are as defined above for Formula (I).

In some embodiments, the compound of Formula (I) is a compound describedin the Examples section.

Preparation of Compounds

Certain compounds of the invention can be prepared following methodologyas described in the Examples section of this document. In addition, thesyntheses of certain intermediate compounds that are useful in thepreparation of compounds of the invention are also described.

B. Pharmaceutical Compositions

In addition to the compounds provided above, compositions for modulatingC5a activity in humans and animals will typically contain apharmaceutical carrier or diluent.

The term “composition” as used herein is intended to encompass a productcomprising the specified ingredients in the specified amounts, as wellas any product which results, directly or indirectly, from combinationof the specified ingredients in the specified amounts. By“pharmaceutically acceptable” it is meant the carrier, diluent orexcipient must be compatible with the other ingredients of theformulation and not deleterious to the recipient thereof.

The pharmaceutical compositions for the administration of the compoundsof this invention may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacyand drug delivery. All methods include the step of bringing the activeingredient into association with the carrier which constitutes one ormore accessory ingredients. In general, the pharmaceutical compositionsare prepared by uniformly and intimately bringing the active ingredientinto association with a liquid carrier or a finely divided solid carrieror both, and then, if necessary, shaping the product into the desiredformulation. In the pharmaceutical composition the active objectcompound is included in an amount sufficient to produce the desiredeffect upon the process or condition of diseases.

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions and self emulsifications as described in U.S. PatentApplication 2002-0012680, hard or soft capsules, syrups, elixirs,solutions, buccal patch, oral gel, chewing gum, chewable tablets,effervescent powder and effervescent tablets. Compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions and such compositions maycontain one or more agents selected from the group consisting ofsweetening agents, flavoring agents, coloring agents, antioxidants andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations. Tablets contain the active ingredient inadmixture with non-toxic pharmaceutically acceptable excipients whichare suitable for the manufacture of tablets. These excipients may be forexample, inert diluents, such as cellulose, silicon dioxide, aluminumoxide, calcium carbonate, sodium carbonate, glucose, mannitol, sorbitol,lactose, calcium phosphate or sodium phosphate; granulating anddisintegrating agents, for example, corn starch, or alginic acid;binding agents, for example PVP, cellulose, PEG, starch, gelatin oracacia, and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated,enterically or otherwise, by known techniques to delay disintegrationand absorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate may beemployed. They may also be coated by the techniques described in theU.S. Pat. Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotictherapeutic tablets for control release.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin, or olive oil.Additionally, emulsions can be prepared with a non-water miscibleingredient such as oils and stabilized with surfactants such asmono-diglycerides, PEG esters and the like.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxy-propylmethylcellulose,sodium alginate, polyvinyl-pyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxy-ethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, may also be present.

The pharmaceutical compositions of the invention may also be in the formof oil-in-water emulsions. The oily phase may be a vegetable oil, forexample olive oil or arachis oil, or a mineral oil, for example liquidparaffin or mixtures of these. Suitable emulsifying agents may benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitolanhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions may also containsweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavoring and coloringagents. Oral solutions can be prepared in combination with, for example,cyclodextrin, PEG and surfactants.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, for example as a solution in 1,3-butane diol. Among theacceptable vehicles and solvents that may be employed are water,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

The compounds of the present invention may also be administered in theform of suppositories for rectal administration of the drug. Thesecompositions can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug.

Such materials include cocoa butter and polyethylene glycols.Additionally, the compounds can be administered via ocular delivery bymeans of solutions or ointments. Still further, transdermal delivery ofthe subject compounds can be accomplished by means of iontophoreticpatches and the like. For topical use, creams, ointments, jellies,solutions or suspensions, etc., containing the compounds of the presentinvention are employed. As used herein, topical application is alsomeant to include the use of mouth washes and gargles.

The compounds of this invention may also be coupled a carrier that is asuitable polymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxy-propyl-methacrylamide-phenol,polyhydroxyethyl-aspartamide-phenol, or polyethyleneoxide-polylysinesubstituted with palmitoyl residues. Furthermore, the compounds of theinvention may be coupled to a carrier that is a class of biodegradablepolymers useful in achieving controlled release of a drug, for examplepolylactic acid, polyglycolic acid, copolymers of polylactic andpolyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross linked or amphipathic block copolymers of hydrogels. Polymers andsemipermeable polymer matrices may be formed into shaped articles, suchas valves, stents, tubing, prostheses and the like. In one embodiment ofthe invention, the compound of the invention is coupled to a polymer orsemipermeable polymer matrix that is formed as a stent or stent-graftdevice.

The pharmaceutical compositions of the present disclosure may beformulated with one or more additional therapeutic agents. The one ormore additional therapeutic agents can include corticosteroids,steroids, immunosuppressants, or CD 20 inhibitors. In some embodiments,the one or more additional therapeutic agents include obinutuzumab,rituximab, ocrelizumab, cyclophosphamide, prednisone, hydrocortisone,hydrocortisone acetate, cortisone acetate, tixocortol pivalate,prednisolone, methylprednisolone, triamcinolone acetonide, triamcinolonealcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide,fluocinolone acetonide, halcinonide, betamethasone, betamethasone sodiumphosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone,hydrocortisone-17-valerate, halometasone, alclometasone dipropionate,beclomethasone, betamethasone valerate, betamethasone dipropionate,prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate,fluocortolone caproate, fluocortolone pivalate, fluprednidene acetate,hydrocortisone-17-butyrate, hydrocortisone-17-aceponate,hydrocortisone-17-buteprate, ciclesonide and prednicarbate. Furtherdiscussions of combination therapy are included in the “Methods of Use”section of this application.

C. Methods of Use

The compounds of the invention may be used as agonists, (preferably)antagonists, partial agonists, inverse agonists, of C5a receptors in avariety of contexts, both in vitro and in vivo. In one embodiment, thecompounds of the invention are C5aR antagonist that can be used toinhibit the binding of C5a receptor ligand (e.g., C5a) to C5a receptorin vitro or in vivo. In general, such methods comprise the step ofcontacting a C5a receptor with a sufficient amount of one or more C5areceptor modulators as provided herein, in the presence of C5a receptorligand in aqueous solution and under conditions otherwise suitable forbinding of the ligand to C5a receptor. The C5a receptor may be presentin suspension (e.g., in an isolated membrane or cell preparation), in acultured or isolated cell, or in a tissue or organ.

Preferably, the amount of C5a receptor modulator contacted with thereceptor should be sufficient to inhibit C5a binding to C5a receptor invitro as measured, for example, using a radioligand binding assay,calcium mobilization assay, or chemotaxis assay as described herein.

In one embodiment of the invention, the C5a modulators of the inventionare used to modulate, preferably inhibit, the signal-transducingactivity of a C5a receptor, for example, by contacting one or morecompound(s) of the invention with a C5a receptor (either in vitro or invivo) under conditions suitable for binding of the modulator(s) to thereceptor. The receptor may be present in solution or suspension, in acultured or isolated cell preparation or within a patient. Anymodulation of the signal transducing activity may be assessed bydetecting an effect on calcium ion calcium mobilization or by detectingan effect on C5a receptor-mediated cellular chemotaxis. In general, aneffective amount of C5a modulator(s) is an amount sufficient to modulateC5a receptor signal transducing activity in vitro within a calciummobilization assay or C5a receptor-mediated cellular chemotaxis within amigration assay.

When compounds of the invention are used to inhibit C5areceptor-mediated cellular chemotaxis, preferably leukocyte (e.g.,neutrophil) chemotaxis, in an in vitro chemotaxis assay, such methodscomprise contacting white blood cells (particularly primate white bloodcells, especially human white blood cells) with one or more compounds ofthe invention. Preferably the concentration is sufficient to inhibitchemotaxis of white blood cells in an in vitro chemotaxis assay, so thatthe levels of chemotaxis observed in a control assay are significantlyhigher, as described above, than the levels observed in an assay towhich a compound of the invention has been added.

In another embodiment, the compounds of the present invention furthercan be used for treating patients suffering from conditions that areresponsive to C5a receptor modulation. As used herein, the term“treating” or “treatment” encompasses both disease-modifying treatmentand symptomatic treatment, either of which may be prophylactic (i.e.,before the onset of symptoms, in order to prevent, delay or reduce theseverity of symptoms) or therapeutic (i.e., after the onset of symptoms,in order to reduce the severity and/or duration of symptoms). As usedherein, a condition is considered “responsive to C5a receptormodulation” if modulation of C5a receptor activity results in thereduction of inappropriate activity of a C5a receptor. As used herein,the term “patients” include primates (especially humans), domesticatedcompanion animals (such as dogs, cats, horses, and the like) andlivestock (such as cattle, pigs, sheep, and the like), with dosages asdescribed herein.

Conditions that can be Treated by C5a Modulation:

Autoimmune Disorders—

e.g., Rheumatoid arthritis, systemic lupus erythematosus, Guillain-Barresyndrome, pancreatitis, lupus nephritis, lupus glomerulonephritis,psoriasis, Crohn's disease, vasculitis, irritable bowel syndrome,dermatomyositis, multiple sclerosis, bronchial asthma, dense depositdisease, pemphigus, pemphigoid, scleroderma, myasthenia gravis,autoimmune hemolytic and thrombocytopenic states, Goodpasture's syndrome(and associated glomerulonephritis and pulmonary hemorrhage),C3-glomerulopathy, C3-glomerulonephritis, membranoproliferativeglomerulonephritis, Kawasaki disease, IGs nephropathy, immunovasculitis,tissue graft rejection, graft versus host disease, hyperacute rejectionof transplanted organs; and the like.

Inflammatory Disorders and Related Conditions—

e.g., Neutropenia, sepsis, septic shock, Alzheimer's disease, multiplesclerosis, neutrophilia, stroke, inflammatory bowel disease (IBD),inflammation associated with severe burns, lung injury, andischemia-reperfusion injury, osteoarthritis, as well as acute (adult)respiratory distress syndrome (ARDS), chronic pulmonary obstructivedisorder (COPD), systemic inflammatory response syndrome (SIRS), atopicdermatitis, psoriasis, chronic urticaria and multiple organ dysfunctionsyndrome (MODS) Hemolytic uremic syndrome, atypical hemolytic uremicsyndrome (aHUS). Also included are pathologic sequellae associated withinsulin-dependent diabetes mellitus (including diabetic retinopathy),lupus nephropathy, Heyman nephritis, membranous nephritis and otherforms of glomerulonephritis, contact sensitivity responses, andinflammation resulting from contact of blood with artificial surfacesthat can cause complement activation, as occurs, for example, duringextracorporeal circulation of blood (e.g., during hemodialysis or via aheart-lung machine, for example, in association with vascular surgerysuch as coronary artery bypass grafting or heart valve replacement), orin association with contact with other artificial vessel or containersurfaces (e.g., ventricular assist devices, artificial heart machines,transfusion tubing, blood storage bags, plasmapheresis,plateletpheresis, and the like). Also included are diseases related toischemia/reperfusion injury, such as those resulting from transplants,including solid organ transplant, and syndromes such as ischemicreperfusion injury, ischemic colitis and cardiac ischemia. Compounds ofthe instant invention may also be useful in the treatment of age-relatedmacular degeneration (Hageman et al, P.N.A.S. 102: 7227-7232, 2005).

Cardiovascular and Cerebrovascular Disorders—

e.g., myocardial infarction, coronary thrombosis, vascular occlusion,post-surgical vascular reocclusion, atherosclerosis, traumatic centralnervous system injury, and ischemic heart disease. In one embodiment, aneffective amount of a compound of the invention may be administered to apatient at risk for myocardial infarction or thrombosis (i.e., a patientwho has one or more recognized risk factor for myocardial infarction orthrombosis, such as, but not limited to, obesity, smoking, high bloodpressure, hypercholesterolemia, previous or genetic history ofmyocardial infarction or thrombosis) in order reduce the risk ofmyocardial infarction or thrombosis.

Oncologic Diseases or Disorders—

e.g., melanoma, lung cancer, lymphoma, sarcoma, carcinoma, fibrosarcoma,liposarcoma, chondrosarcoma, osteogenic sarcoma, angiosarcoma,lymphangiosarcoma, synovioma, mesothelioma, meningioma, leukemia,lymphoma, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, papillary carcinoma,cystadenocarcinoma, bronchogenic carcinoma, renal cell carcinoma,hepatocellular carcinoma, transitional cell carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, wilm's tumor, pleomorphic adenoma, livercell papilloma, renal tubular adenoma, cystadenoma, papilloma, adenoma,leiomyoma, rhabdomyoma, hemangioma, lymphangioma, osteoma, chondroma,lipoma and fibroma.

Diseases of Vasculitis—

Vasculitic dseases are characterized by inflammation of the vessels.Infliltration of leukocytes leads to destruction of the vessel walls,and the complement pathway is believed to play a major role ininitiating leukocyte migration as well as the resultant damagemanifested at the site of inflammation (Vasculitis, Second Edition,Edited by Ball and Bridges, Oxford University Press, pp 47-53, 2008).The compounds provided in the present invention can be used to treatleukoclastic vasculitis, Anti-neutrophil cytoplasmic antibody (ANCA)associated vasculitis, immune vasculitis Wegener's granulomatosis,microscopic polyangiitis, Churg-Strauss syndrome, Henoch-Schonleinpurpura, polyateritis nodosa, Rapidly Progressive Glomerulonephritis(RPGN), cryoglobulinaemia, giant cell arteritis (GCA), Behcet's diseaseand Takayasu's arteritis (TAK).

HIV infection and AIDS—

C5a receptor modulators provided herein may be used to inhibit HIVinfection, delay AIDS progression or decrease the severity of symptomsor HIV infection and AIDS.

Neurodegenerative Disorders and Related Diseases—

Within further aspects, C5a antagonists provided herein may be used totreat Alzheimer's disease, multiple sclerosis, and cognitive functiondecline associated with cardiopulmonary bypass surgery and relatedprocedures.

In one embodiment of the invention, the compounds of the invention canbe used for the treatment of diseases selected from the group consistingof sepsis (and associated disorders), COPD, rheumatoid arthritis, lupusnephritis and multiple sclerosis.

Treatment methods provided herein include, in general, administration toa patient an effective amount of one or more compounds provided herein.Suitable patients include those patients suffering from or susceptibleto (i.e., prophylactic treatment) a disorder or disease identifiedherein. Typical patients for treatment as described herein includemammals, particularly primates, especially humans. Other suitablepatients include domesticated companion animals such as a dog, cat,horse, and the like, or a livestock animal such as cattle, pig, sheepand the like.

In general, treatment methods provided herein comprise administering toa patient an effective amount of a compound one or more compoundsprovided herein. In a preferred embodiment, the compound(s) of theinvention are preferably administered to a patient (e.g., a human)orally or topically. The effective amount may be an amount sufficient tomodulate C5a receptor activity and/or an amount sufficient to reduce oralleviate the symptoms presented by the patient. Preferably, the amountadministered is sufficient to yield a plasma concentration of thecompound (or its active metabolite, if the compound is a pro-drug) highenough to detectably inhibit white blood cell (e.g., neutrophil)chemotaxis in vitro. Treatment regimens may vary depending on thecompound used and the particular condition to be treated; for treatmentof most disorders, a frequency of administration of 4 times daily orless is preferred. In general, a dosage regimen of 2 times daily is morepreferred, with once a day dosing particularly preferred. It will beunderstood, however, that the specific dose level and treatment regimenfor any particular patient will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, sex, diet, time of administration, route ofadministration, rate of excretion, drug combination (i.e., other drugsbeing administered to the patient) and the severity of the particulardisease undergoing therapy, as well as the judgment of the prescribingmedical practitioner. In general, the use of the minimum dose sufficientto provide effective therapy is preferred. Patients may generally bemonitored for therapeutic effectiveness using medical or veterinarycriteria suitable for the condition being treated or prevented.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment orpreventions of conditions involving pathogenic C5a activity (about 0.5mg to about 7 g per human patient per day). The amount of activeingredient that may be combined with the carrier materials to produce asingle dosage form will vary depending upon the host treated and theparticular mode of administration. Dosage unit forms will generallycontain between from about 1 mg to about 500 mg of an active ingredient.For compounds administered orally, transdermally, intravenously, orsubcutaneously, it is preferred that sufficient amount of the compoundbe administered to achieve a serum concentration of 5 ng(nanograms)/mL-10 μg (micrograms)/mL serum, more preferably sufficientcompound to achieve a serum concentration of 20 ng-1 μg/ml serum shouldbe administered, most preferably sufficient compound to achieve a serumconcentration of 50 ng/ml-200 ng/ml serum should be administered. Fordirect injection into the synovium (for the treatment of arthritis)sufficient compounds should be administered to achieve a localconcentration of approximately 1 micromolar.

Frequency of dosage may also vary depending on the compound used and theparticular disease treated. However, for treatment of most disorders, adosage regimen of 4 times daily, three times daily, or less ispreferred, with a dosage regimen of once daily or 2 times daily beingparticularly preferred. It will be understood, however, that thespecific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, route of administration, and rate of excretion, drugcombination (i.e., other drugs being administered to the patient), theseverity of the particular disease undergoing therapy, and otherfactors, including the judgment of the prescribing medical practitioner.

Combination Therapy

The presently disclosed compounds may be used in combination with one ormore additional therapeutic agents that are used in the treatment,prevention, suppression or amelioration of the diseases or conditionsfor which compounds and compositions of the present invention areuseful. Such one or more additional therapeutic agents may beadministered, by a route and in an amount commonly used therefor,contemporaneously or sequentially with a compound or composition of thepresent invention. When a compound or composition of the presentinvention is used contemporaneously with one or more other drugs, apharmaceutical composition containing such other drugs in addition tothe compound or composition of the present invention is preferred.Accordingly, the pharmaceutical compositions of the present inventioninclude those that also contain one or more other active ingredients ortherapeutic agents, in addition to a compound or composition of thepresent invention.

Examples of the one or more additional therapeutic agents that may becombined with a compound or composition of the present invention, eitheradministered separately or in the same pharmaceutical compositions,include, but are not limited to: (a) VLA-4 antagonists, (b) steroids andcorticosteroids, such as beclomethasone, betamethasone (includingbetamethasone sodium phosphate, betamethasone valerate, betamethasonedipropionate) prednisone, prenisolone, methylprednisolone, mometasone,dexamethasone (including dexamethasone sodium phosphate), fluticasone,cortisone (including cortisone acetate) hydrocortisone (includinghydrocortisone acetate, hydrocortisone-17-valerate,hydrocortisone-17-butyrate, hydrocortisone-17-aceponate,hydrocortisone-17-buteprate), budesonide, desonide, fluocinonide(including fluocinolone acetonide), triamcinolone (includingtriamcinolone acetonide and triamcinolone alcohol), tixocortol(including tixocortol pivalate) fluocortolone (including fluocortolonecaproate and fluocortolone pivalate), amcinonide, halcinonide,halometasone, fluprednidene acetate, salmeterol, salmeterol, salbutamol,ciclesonide, formeterol, alclometasone (including alclometasonedipropionate), prednicarbate, clobetasone (includingclobetasone-17-butrate), clobetasol (includingclobetasol-17-propionate); (c) immunosuppressants such as cyclosporine(cyclosporine A, Sandimmune®, Neoral®), tacrolirnus (FK-506, Prograf®),rapamycin (sirolimus, Rapamune®) and other FK-506 typeimmunosuppressants, and rnycophenolate, e.g., mycophenolate mofetil(CellCept8); (d) antihistamines (H1-histamine antagonists) such asbromopheniramine, chlorpheniramine, dexchloipheniramine, triprolidine,clemastine, diphenhydramine, diphenylpyraline, tripelennamine,hydroxyzine, methdilazine, promethazine, trimeprazine, azatadine,cyproheptadine, antazoline, pheniramine pyrilamine, astemizole,terfenadine, loratadine, cetirizine, fexofenadine,descarboethoxyloratadine, and the like; (e) non steroidal antiasthmatics (e.g., terbutaline, metaproterenol, fenoterol, isoetharine,albuterol, bitolterol and pirbuterol), theophylline, cromolyn sodium,atropine, ipratropium bromide, leukotriene antagonists (e.g.,zafmlukast, montelukast, pranlukast, iralukast, pobilukast andSKB-106,203), leukotriene biosynthesis inhibitors (zileuton, BAY-1005);(f) non steroidal anti-inflammatory agents (NSAIDs) such as propionicacid derivatives (e.g., alminoprofen, benoxaprofen, bucloxic acid,carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen, ibuprofen,indoprofen, ketoprofen, rniroprofen, naproxen, oxaprozin, pirprofen,pranoprofen, suprofen, tiaprofenic acid and tioxaprofen), acetic acidderivatives (e.g., indomethacin, acemetacin, alclofenac, clidanac,diclofenac, fenclofenac, fenclozic acid, fentiazac, furofenac, ibufenac,isoxepac, oxpinac, sulindac, tiopinac, tolmetin, zidometacin andzomepirac), fenamic acid derivatives (e.g., flufenamic acid,meclofenamic acid, mefenamic acid, niflumic acid and tolfenamic acid),biphenylcarboxylic acid derivatives (e.g., diflunisal and flufenisal),oxicams (e.g., isoxicam, piroxicam, sudoxicam and tenoxican),salicylates (e.g., acetyl salicylic acid and sulfasalazine) and thepyrazolones (e.g., apazone, bezpiperylon, feprazone, mofebutazone,oxyphenbutazone and phenylbutazone); (g) cyclooxygenase-2 (COX-2)inhibitors such as celecoxib (Celebrex®) and rofecoxib (Vioxx®); (h)inhibitors of phosphodiesterase type IV (PDE IV); (i) gold compoundssuch as auranofin and aurothioglucose, (j) etanercept (Enbrel®), (k)cyclophosphamide, (1) antibody therapies such as orthoclone (OKT3),daclizumab (Zenapax®), basiliximab (Simulect®) and infliximab(Remicade®), (m) antibody therapies targeting CD20 such as obinutuzumab,rituximab, or ocrelizumab; (n) chemotherapeutic agents suchanthracyclines (e.g., daunorubicin (daunomycin; rubidomycin),doxorubicin, epirubicin, idarubicin, and valrubicin), mitoxantrone, andpixantrone; platinum-based agents (e.g., cisplatin, carboplatin,oxaliplatin, satraplatin, picoplatin, nedaplatin, triplatin, andlipoplatin); tamoxifen and metabolites thereof such as4-hydroxytamoxifen (afimoxifene) and N-desmethyl-4-hydroxytamoxifen(endoxifen); taxanes such as paclitaxel (taxol) and docetaxel;alkylating agents (e.g., nitrogen mustards such as mechlorethamine(HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), andchlorambucil); ethylenimines and methylmelamines (e.g.,hexamethylmelamine, thiotepa, alkyl sulphonates such as busulfan,nitrosoureas such as carmustine (BCNU), lomustine (CCNLJ), semustine(methyl-CCN-U), and streptozoein (streptozotocin), and triazenes such asdecarbazine (DTIC; dimethyltriazenoimidazolecarboxamide));antimetabolites (e.g., folic acid analogues such as methotrexate(amethopterin), pyrimidine analogues such as fluorouracil(5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR), andcytarabine (cytosine arabinoside), and purine analogues and relatedinhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine(6-thioguanine; 6-TG), and pentostatin (2′-deoxycofonnycin)); (o) otherantagonists of the chemokine receptors, especially CXCR2, CXCR3, CCR2,CCR3, CCR4, CCR7, CX3CR1 and CXCR6.

The disease or disorder being treated will determine which additionaltherapeutic agent or therapeutic agents are most appropriatelyadministered in combination with the compounds of the presentinvention—such determination can be made by a person of skill in theart.

The weight ratio of the compound of the present invention to the secondactive ingredient may be varied and will depend upon the effective doseof each ingredient. Generally, an effective dose of each will be used.Thus, for example, when a compound of the present invention is combinedwith an NSAID the weight ratio of the compound of the present inventionto the NSAID will generally range from about 1000:1 to about 1:1000,preferably about 200:1 to about 1:200. Combinations of a compound of thepresent invention and other active ingredients will generally also bewithin the aforementioned range, but in each case, an effective dose ofeach active ingredient should be used.

Non-Pharmaceutical Applications

In another aspect of the invention, the compounds of the invention canbe used in a variety of non-pharmaceutical in vitro and in vivoapplication. For example, the compounds of the invention may be labeledand used as probes for the detection and localization of C5a receptor(cell preparations or tissue sections samples). The compounds of theinvention may also be used as positive controls in assays for C5areceptor activity, i.e., as standards for determining the ability of acandidate agent to bind to C5a receptor, or as radiotracers for positronemission tomography (PET) imaging or for single photon emissioncomputerized tomography (SPECT). Such methods can be used tocharacterize C5a receptors in living subjects. For example, a C5areceptor modulator may be labeled using any of a variety of well knowntechniques (e.g., radiolabeled with a radionuclide such as tritium), andincubated with a sample for a suitable incubation time (e.g., determinedby first assaying a time course of binding). Following incubation,unbound compound is removed (e.g., by washing), and bound compounddetected using any method suitable for the label employed (e.g.,autoradiography or scintillation counting for radiolabeled compounds;spectroscopic methods may be used to detect luminescent groups andfluorescent groups). As a control, a matched sample containing labeledcompound and a greater (e.g., 10-fold greater) amount of unlabeledcompound may be processed in the same manner. A greater amount ofdetectable label remaining in the test sample than in the controlindicates the presence of C5a receptor in the sample. Detection assays,including receptor autoradiography (receptor mapping) of C5a receptor incultured cells or tissue samples may be performed as described by Kuharin sections 8.1.1 to 8.1.9 of Current Protocols in Pharmacology (1998)John Wiley & Sons, New York.

The compounds provided herein may also be used within a variety of wellknown cell separation methods. For example, modulators may be linked tothe interior surface of a tissue culture plate or other support, for useas affinity ligands for immobilizing and thereby isolating, C5areceptors (e.g., isolating receptor-expressing cells) in vitro. In onepreferred application, a modulator linked to a fluorescent marker, suchas fluorescein, is contacted with the cells, which are then analyzed (orisolated) by fluorescence activated cell sorting (FACS).

III. Examples

The following examples are offered to illustrate, but not to limit theclaimed invention.

Reagents and solvents used below can be obtained from commercial sourcessuch as Aldrich Chemical Co. (Milwaukee, Wis., USA). ¹H-NMR spectra wererecorded on a Varian Mercury 400 MHz NMR spectrometer. Significant peaksare provided relative to TMS and are tabulated in the order:multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m,multiplet) and number of protons. Mass spectrometry results are reportedas the ratio of mass over charge, followed by the relative abundance ofeach ion (in parenthesis). In the examples, a single m/e value isreported for the M+H (or, as noted, M−H) ion containing the most commonatomic isotopes. Isotope patterns correspond to the expected formula inall cases. Electrospray ionization (ESI) mass spectrometry analysis wasconducted on a Hewlett-Packard MSD electrospray mass spectrometer usingthe HP1100 HPLC for sample delivery. Normally the analyte was dissolvedin methanol at 0.1 mg/mL and 1 microliter was infused with the deliverysolvent into the mass spectrometer, which scanned from 100 to 1500daltons. All compounds could be analyzed in the positive ESI mode, usingacetonitrile/water with 1% formic acid as the delivery solvent. Thecompounds provided below could also be analyzed in the negative ESImode, using 2 mM NH₄OAc in acetonitrile/water as delivery system.

The following abbreviations are used in the Examples and throughout thedescription of the invention:

EtOH: Ethanol

EtONa: Sodium ethoxide

THF: Tetrahydrofuran

TLC: Thin layer chromatography

MeOH: Methanol

Compounds within the scope of this invention can be synthesized asdescribed below, using a variety of reactions known to the skilledartisan. One skilled in the art will also recognize that alternativemethods may be employed to synthesize the target compounds of thisinvention, and that the approaches described within the body of thisdocument are not exhaustive, but do provide broadly applicable andpractical routes to compounds of interest.

Certain molecules claimed in this patent can exist in differentenantiomeric and diastereomeric forms and all such variants of thesecompounds are claimed.

The detailed description of the experimental procedures used tosynthesize key compounds in this text lead to molecules that aredescribed by the physical data identifying them as well as by thestructural depictions associated with them.

Those skilled in the art will also recognize that during standard workup procedures in organic chemistry, acids and bases are frequently used.Salts of the parent compounds are sometimes produced, if they possessthe necessary intrinsic acidity or basicity, during the experimentalprocedures described within this patent.

Example 1 Synthesis of intermediate6-fluoro-7-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole

Step a: Vinylmagnesium bromide solution in THF (1.0 M, 70 mL, 70 mmol)was added to a solution of 4-bromo-2-fluoro-6-nitroanisole (5.0 g, 20mmol) in anhydrous THF (70 mL) under N₂ at −50° C. The reaction mixturewas stirred at the same temperature and allowed to warm to −30° C. over1.5 h. The reaction mixture was quenched with saturated aqueous NH₄Clsolution and allowed to warm up to room temperature over 1 h. Thereaction mixture was diluted with EtOAc, washed with brine and driedover Na₂SO₄. The solvent was removed under reduced pressure and theresidue was purified by silica gel flash chromatography (0 to 100% EtOAcin hexanes) to yield 4-bromo-6-fluoro-7-methoxy-1H-indole. MS: (ES) m/zcalculated for C₉H₈BrFNO [M+H]⁺ 243.9, found 243.9.

Step b: To a suspension of 4-bromo-6-fluoro-7-methoxy-1H-indole (900 mg,3.68 mmol), bis(pinacolato)diboron (1.21 g, 4.8 mmol) and KOAc (1.08 g,11 mmol) in dioxane (16 mL) was added Pd(dppf)Cl₂ complex withdichloromethane (400 mg, 0.49 mmol). The reaction mixture was degassedwith N₂ for 2 min and stirred at 100° C. for 2 h. The reaction mixturewas diluted with EtOAc and filtered through Celite. The solvent wasremoved under reduced pressure and the residue was purified by silicagel flash chromatography (0 to 100% EtOAc in hexanes) to give6-fluoro-7-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole.MS: (ES) m/z calculated for C₁₅H₂₀BFNO₃ [M+H]⁺ 292.1, found 292.1.

Example 2 Synthesis of4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-6-fluoro-7-methoxy-1H-indole

Step a: To a 250 mL flask containing 90 mL of concentrated hydrochloricacid with magnetic stirring was added 2,6-diethylaniline (10 g, 67mmol). The resulting mixture was stirred for 30 min and cooled with anice/salt bath until the internal temperature reached −5° C. A solutionof sodium nitrite (5.5 g, 80 mmol) in water (60 mL) was added slowly tothe above mixture while maintaining the internal temperature below 5° C.

Separately, tin(II) chloride dihydrate (31.6 g, 140 mmol) was added to a500 mL 3-neck round bottom flask containing concentrated hydrochloricacid (60 mL) with mechanical stirring. The resulting solution was thencooled with an ice bath.

The diazonium slurry was then filtered into the 500 mL flask containingthe vigorously stirred and cold tin chloride solution. After 90 min, thereaction mixture was transferred to a 500 mL Erlenmeyer flask and theflask was rinsed with water (20 mL) and chloroform (8 mL). The combinedmixture was stirred overnight at room temperature. The liquid wasdecanted to give a wet solid. The solid was dried in vacuo for one dayand then transferred to a 500 mL 3-neck round bottom flask equipped witha mechanical stirrer and stirred with ether (180 mL). The resultingmixture was cooled in an ice bath and 10 N NaOH solution (30 mL) wasadded slowly to the mixture while maintaining the internal temperaturebelow 12° C. After the addition, the mixture was allowed to stand for 2h on ice. The ether layer was decanted into a 500 mL flask and a streamof hydrogen chloride gas was bubbled into the stirred ether solution.The resulting precipitate was collected by filtration to afford(2,6-diethylphenyl)hydrazine hydrochloride. MS: (ES) m/z calculated forC₁₀H₁₇N₂[M+H]⁺ 165.1, found 165.1.

Step b: Pyridine (4 mL, 49.5 mmol) was added to a mixture of(2,6-diethylphenyl)hydrazine hydrochloride (5 g, 24.91 mmol), tert-butyl4-cyano-2,2-dimethyl-3-oxopyrrolidine-1-carboxylate (5 g, 20.98 mmol)and EtOH (60 mL) in a 250 mL round bottom flask under magnetic stirring.The resulting mixture was stirred at 70° C. for 24 h. The solvent wasremoved under reduced pressure and the residue was diluted with EtOAcand washed with aqueous citric acid solution, aqueous NaHCO₃ solution,brine, and dried over MgSO₄. The solvent was removed under reducedpressure and the residue was crystallized from cyclohexane to givetert-butyl3-amino-2-(2,6-diethylphenyl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate.MS: (ES) m/z calculated for C₂₂H₃₃N₄O₂ [M+H]⁺ 385.2, found 385.2.

Tert-butyl nitrite (0.5 mL, 3.8 mmol) was added slowly at roomtemperature to a mixture of tert-butyl3-amino-2-(2,6-diethylphenyl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate(1 g, 2.6 mmol), diiodomethane (1.5 mL, 18.6 mmol) and MeCN (15 mL) in a100 mL round bottom flask under magnetic stirring. The resulting mixturewas stirred at 45° C. for 3 h before it was diluted with toluene, washedwith saturated NH₄Cl solution/NH₄OH (3:1), brine, and dried over MgSO₄.The solvent was removed under reduced pressure and the residue waspurified by silica gel flash chromatography (2 to 25% EtOAc in hexanes)to give tert-butyl2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate.MS: (ES) m/z calculated for C₂₂H₃₁IN₃O₂[M+H]⁺ 496.1, found 496.2.

Step c: The above tert-butyl2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylatewas dissolved in dichloromethane (10 mL) and charged with HCl in dioxane(4 N, 5 mL). The resulting mixture was stirred at room temperature for12 h. Upon completion, the solvent was evaporated in vacuo to give2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolehydrochloride. MS: (ES) m/z calculated for C₁₇H₂₃IN₃ [M+H]⁺ 396.1, found396.2.

N,N-diisopropylethylamine (0.3 mL, 1.73 mmol) was added to a suspensionof2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolehydrochloride (680 mg, 1.57 mmol), and2,4-bis(trifluoromethyl)benzaldehyde (800 mg, 3.3 mmol) in1,2-dichloroethane (10 mL) under magnetic stirring. After stirring atroom temperature for 10 min, NaBH(OAc)₃ (800 mg, 3.77 mmol) was added inportions. The resulting mixture was stirred at 45° C. for 2 h. Aftercooling to room temperature, the reaction mixture was diluted withEtOAc, washed with aqueous NaHCO₃ solution, brine and dried over MgSO₄.The solvent was removed under reduced pressure and the residue waspurified by silica gel flash chromatography (2 to 25% EtOAc in hexanes)to give5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole.MS: (ES) m/z calculated for C₂₆H₂₇F₆₁N₃ [M+H]⁺ 622.1, found 622.1.

Step d: To a suspension of5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole(250 mg, 0.40 mmol),6-fluoro-7-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole(100 mg, 0.34 mmol) and K₂CO₃ (445 mg, 1.81 mmol) in dioxane (6 mL) andwater (1 mL) was added Pd(dppf)Cl₂ complex with dichloromethane (50 mg,0.06 mmol). The reaction mixture was degassed with N₂ for 2 min andstirred under N₂ at 100° C. for 2.5 h. The reaction mixture was dilutedwith EtOAc, washed with aqueous NaHCO₃ and dried over Na₂SO₄. Thesolvent was removed under reduced pressure and the residue was purifiedby silica gel flash chromatography (3 to 35% EtOAc in hexanes) followedby HPLC (MeCN/H₂O, with 1% TFA) to give4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-6-fluoro-7-methoxy-1H-indole.¹H NMR (400 MHz, CDCl₃) δ 8.37 (br s, 1H), 8.19 (d, J=12 Hz, 1H), 7.86(s, 1H), 7.76 (d, J=12 Hz, 1H), 7.07-7.33 (m, 4H), 6.41 (dd, J=2.2, 3.2Hz, 1H), 6.33 (d, J=13.8 Hz, 1H), 4.14 (s, 2H), 4.01 (s, 3H), 3.70 (s,2H), 2.23-2.37 (m, 4H), 1.55 (s, 6H), 1.02 (t, J=7.6 Hz, 6H). MS: (ES)m/z calculated for C₃₅H₃₄F₇N₄O [M+H]⁺ 659.2, found 659.2.

Example 3 Synthesis of3-chloro-4-(2-(2,6-diethylphenyl)-6,6-dimethyl-5-(2-(trifluoromethyl)benzyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole

Step a: To a suspension of 4-bromo-7-fluoro-1H-indole (1.00 g, 4.67mmol), bis(pinacolato)diboron (1.31 g, 5.14 mmol) and KOAc (1.15 g, 11.7mmol) in dioxane (15 mL) was added Pd(dppf)Cl₂ complex withdichloromethane (416 mg, 0.51 mmol). The reaction mixture was degassedwith N₂ for 2 min and stirred at 100° C. for 2 h. The reaction mixturewas cooled to room temperature, diluted with EtOAc, and filtered throughCelite. The solvent was removed under reduced pressure and the residuewas purified by silica gel flash chromatography (0 to 30% EtOAc inhexanes) to give7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. MS:(ES) m/z calculated for C₁₄H₁₈BFNO₂ [M+H]⁺ 262.1, found 262.1.

Step b: To a suspension of tert-butyl2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate(540 mg, 1.09 mmol),7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (350mg, 1.34 mmol), and K₂CO₃ (830 mg, 6.78 mmol) in dioxane (10 mL) andwater (2 mL) was added Pd(dppf)Cl₂ complex with dichloromethane (200 mg,0.32 mmol). The reaction mixture was degassed with N₂ for 2 min andstirred under N₂ at 100° C. for 2 h. The reaction mixture was cooled toroom temperature, diluted with EtOAc, washed with aqueous NaHCO₃, anddried over Na₂SO₄. The solvent was removed under reduced pressure andthe residue was purified by silica gel flash chromatography (5 to 25%EtOAc in hexanes) to give tert-butyl2-(2,6-diethylphenyl)-3-(7-fluoro-1H-indol-4-yl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate.MS: (ES) m/z calculated for C₃₀H₃₆FN₄O₂[M+H]⁺ 503.2, found 503.3.

Step c: The above tert-butyl2-(2,6-diethylphenyl)-3-(7-fluoro-1H-indol-4-yl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate(200 mg, 0.40 mmol) was dissolved in DMF (5 mL) and charged withN-chlorosuccinimide (100 mg, 0.75 mmol). The resulting mixture wasstirred at room temperature for 12 h. The reaction mixture was dilutedwith EtOAc, washed with aqueous Na₂S₂O₃ solution, brine, and dried overMgSO₄. The solvent was removed under reduced pressure and the residuewas purified by silica gel flash chromatography (2 to 25% EtOAc inhexanes) to give tert-butyl3-(3-chloro-7-fluoro-1H-indol-4-yl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylate.MS: (ES) m/z calculated for C₃₀H₃₃ClFN₄O₂[M−H]⁻ 535.2, found 535.2.

The above tert-butyl3-(3-chloro-7-fluoro-1H-indol-4-yl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazole-5(4H)-carboxylatewas dissolved in dichloromethane (6 mL) and charged with HCl in dioxane(4 N, 5 mL). The resulting mixture was stirred at room temperature for12 h. After the reaction was complete, the solvent was evaporated invacuo to give3-chloro-4-(2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indolehydrochloride. MS: (ES) m/z calculated for C₂₅H₂₇ClFN₄ [M+H]⁺ 437.2,found 437.2.

Step d: N,N-diisopropylethylamine (0.2 mL, 1.15 mmol) was added to asuspension of3-chloro-4-(2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indolehydrochloride (75 mg, 0.16 mmol), and 2-trifluoromethylbenzaldehyde (120mg, 0.69 mmol) in 1,2-dichloroethane (6 mL) under magnetic stirring.After stirring at room temperature for 10 min, NaBH(OAc)₃ (150 mg, 0.71mmol) was added to the reaction mixture. The resulting mixture wasstirred at 40° C. for 1 h, then cooled to room temperature, diluted withEtOAc, washed with brine, and dried over MgSO₄. The solvent was removedunder reduced pressure and the residue was purified by preparative TLC(40% EtOAc in hexanes) followed by HPLC (MeCN/H₂O, with 1% TFA) to give3-chloro-4-(2-(2,6-diethylphenyl)-6,6-dimethyl-5-(2-(trifluoromethyl)benzyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole.¹H NMR (400 MHz, CD₃OD) δ 7.92-7.99 (m, 1H), 7.52-7.66 (m, 2H),7.34-7.38 (m, 2H), 7.24-7.29 (m, 2H), 6.94-6.98 (m, 1H), 6.66 (dd,J=8.2, 10.8 Hz, 1H), 6.50 (dd, J=4.5, 8.2 Hz, 1H), 4.88 (br, 1H), 4.09(s, 2H), 3.82 (d, J=11.5 Hz, 1H), 3.43 (d, J=11.5 Hz, 1H), 2.53 (dq,J=7.5, 15 Hz, 1H), 2.37-2.46 (m, 2H), 2.07 (dq, J=7.5, 15 Hz, 1H), 1.54(s, 3H), 1.51 (s, 3H), 1.33 (t, J=7.6 Hz, 3H), 0.76 (t, J=7.6 Hz, 3H).MS: (ES) m/z calculated for C₃₃H₃₂ClF₄N₄[M+H]⁺ 595.2, found 595.2.

Example 4 Synthesis of4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole

A mixture of5-(2,4-bis(trifluoromethyl)benzyl)-3-iodo-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole(200 mg, 0.32 mmol),7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (150mg, 0.57 mmol), K₂CO₃ (276 mg, 2.0 mmol) and Pd(dppf)Cl₂ complex withdichloromethane (60 mg, 0.07 mmol) in dioxane (6 mL) and water (1 mL)was stirred at 100° C. for 5 h under N₂. The mixture was cooled to roomtemperature, diluted with EtOAc, and filtered through a plug of Celite.The filtrate was collected, concentrated in vacuo and the residue waspurified by silica gel flash chromatography (0 to 50% EtOAc in hexanes)to yield4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole.¹H NMR (400 MHz, CDCl₃) δ 8.44 (s, 1H), 8.19 (d, J=8.0 Hz, 1H), 7.86 (s,1H), 7.75 (d, J=8.4 Hz, 1H), 7.22 (m, 2H), 7.07 (d, J=7.6 Hz, 2H), 6.61(m, 1H), 6.47 (m, 2H), 4.15 (s, 2H), 3.71 (s, 2H), 2.37 (m, 2H), 2.22(m, 2H), 1.56 (s, 6H), 1.00 (t, J=7.6 Hz, 6H). MS: (ES) m/z calculatedfor C₃₄H₃₂F₇N₄ [M+H]⁺ 629.2, found 629.2.

Example 5 Synthesis of4-(5-(3,5-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole

Step a: A mixture of2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazolehydrochloride (2.20 g, 5.1 mmol), 3,5-bis(trifluoromethyl)benzaldehyde(1.85 g, 7.6 mmol), NaBH(OAc)₃ (3.24 g, 15.3 mmol), iPrNEt₂ (0.84 mL,5.1 mmol) and acetic acid (0.49 mL, 7.6 mmol) in DCM (30 mL) was stirredat room temperature for 1.5 h. It was then quenched with aqueous NaHCO₃and extracted with EtOAc. The organic layer was separated, dried overNa₂SO₄, concentrated in vacuo, and the residue was purified by silicagel flash chromatography (0 to 30% EtOAc in hexanes) to give5-(3,5-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole.MS: (ES) m/z calculated for C₂₆H₂₇FIN₃ [M+H]⁺ 622.1, found 622.1.

Step b: A mixture of5-(3,5-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole(1.50 g, 2.4 mmol),7-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (817mg, 3.1 mmol), K₂CO₃ (0.830 g, 6.0 mmol) and Pd(dppf)Cl₂ complex withdichloromethane (30 mg, 0.36 mmol) in dioxane (12 mL) and water (1.5 mL)was stirred at 100° C. for 2 h under N₂. The reaction mixture was cooledto room temperature and partitioned between EtOAc and aqueous NaHCO₃.The organic layer was separated, dried over Na₂SO₄, and concentrated invacuo. The residue was purified by silica gel flash chromatography (0 to50% EtOAc in hexanes) followed by HPLC (MeCN/H₂O, with 1% TFA) to give4-(5-(3,5-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole.¹H NMR (400 MHz, CDCl₃) δ 8.48 (s, 1H), 7.92 (s, 2H), 7.14 (s, 1H), 7.24(dd, J=7.6, 7.6 Hz, 1H), 7.20 (d, J=2.8 Hz, 1H), 7.07 (dd, J=7.6, 7.6Hz, 2H), 6.61 (m, 1H), 6.47 (m, 2H), 4.03 (s, 2H), 3.65 (s, 2H), 2.37(m, 2H), 2.22 (m, 2H), 1.56 (s, 6H), 1.00 (t, J=7.6 Hz, 6H). MS: (ES)m/z calculated for C₃₄H₃₂F₇N₄ [M+H]⁺ 629.2, found 629.2.

Example 6 Synthesis of intermediate methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-7-carboxylate

Step a: To a suspension of methyl 4-bromo-1H-indole-7-carboxylate (300mg, 1.18 mmol), bis(pinacolato)diboron (330 mg, 1.30 mmol), and KOAc(290 mg, 2.96 mmol) in dioxane (8 mL) was added Pd(dppf)Cl₂ complex withdichloromethane (100 mg, 0.12 mmol). The reaction mixture was degassedwith N₂ for 2 min and stirred at 100° C. for 1 h. The reaction mixturewas diluted with EtOAc, filtered through Celite. The solvent was removedunder reduced pressure and the residue was purified by silica gel flashchromatography (5 to 30% EtOAc in hexanes) to give methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-7-carboxylate.MS: (ES) m/z calculated for C₁₆H₂₁BNO₄ [M+H]⁺ 302.2, found 302.2.

Example 7 Synthesis of(4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-1H-indol-7-yl)methanol

Step a: A mixture of5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole(100 mg, 0.16 mmol), methyl4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole-7-carboxylate(78 mg, 0.26 mmol), K₂CO₃ (150 mg, 1.1 mmol) and Pd(dppf)Cl₂ complexwith dichloromethane (90 mg, 0.11 mmol) in dioxane (4 mL) and water (0.7mL) was stirred at 100° C. for 2 h under N₂. The reaction mixture wascooled to room temperature and partitioned between EtOAc and aqueousNaHCO₃. The organic layer was separated, dried over Na₂SO₄, andconcentrated in vacuo. The residue was purified by silica gel flashchromatography (0 to 40% EtOAc in hexanes) to give methyl4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-1H-indole-7-carboxylate.MS: (ES) m/z calculated for C₃₆H₃₅F₆N₄O₂[M+H]⁺ 669.3, found 669.3.

Step b: To a solution of methyl4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-1H-indole-7-carboxylate(25 mg, 0.037 mmol) in THF (1.5 mL) at 0° C. was added a solution ofLiAlH₄ in ether (1.0 M, 0.20 mL, 0.20 mmol). After 1 h at 0° C., thereaction mixture was quenched with water and partitioned between EtOAcand aqueous NaHCO₃. The organic layer was separated, dried over Na₂SO₄,and concentrated in vacuo. The residue was purified by HPLC (MeCN/H₂O,with 1% TFA) followed by silica gel flash chromatography (0 to 60% EtOAcin hexanes) to give(4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-1H-indol-7-yl)methanol.¹H NMR (400 MHz, CDCl₃) δ 8.99 (s, 1H), 8.18 (d, J=8.0 Hz, 1H), 7.86 (s,1H), 7.74 (d, J=8.4 Hz, 1H), 7.24 (m, 2H), 7.06 (d, J=7.6 Hz, 2H), 6.70(d, J=7.4 Hz, 1H), 6.49 (d, J=7.4 Hz, 1H), 6.48 (d, J=2.6 Hz, 1H), 4.92(d, J=5.6 Hz, 2H), 4.15 (s, 2H), 3.72 (s, 2H), 2.39 (m, 2H), 2.26 (m,2H), 1.83 (t, J=5.8 Hz, 1H), 1.56 (s, 6H), 1.02 (t, J=7.6, 6H). MS: (ES)m/z calculated for C₃₅H₃₅F₆N₄O [M+H]⁺ 641.3, found 641.3.

Example 8 Synthesis of intermediate3,3,3-trifluoro-2,2-dimethylpropanoyl chloride

A mixture of 3,3,3-trifluoro-2,2-dimethylpropanoic acid (0.312 g, 2.0mmol), oxalyl chloride (0.17 mL, 2.0 mmol) and DMF (2 drops) in DCM (6.7mL) was stirred at room temperature for 30 min. The reaction mixturecontaining 3,3,3-trifluoro-2,2-dimethylpropanoyl chloride was directlyused in the subsequent step without further purification.

Example 9 Synthesis of4-(2-(2,6-diethylphenyl)-6,6-dimethyl-5-(3,3,3-trifluoro-2,2-dimethylpropyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole

Step a: A mixture of4-(2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indolehydrochloride (78 mg, 0.18 mmol), 3,3,3-trifluoro-2,2-dimethylpropanoylchloride (˜2 mmol) and NEt₃ (0.13 mL, 0.89 mmol) in DCM (3 mL) wasstirred for 1 h at room temperature. The mixture was then partitionedbetween EtOAc and aqueous NaHCO₃. The organic layer was separated, driedover Na₂SO₄, and concentrated in vacuo. The residue was purified bysilica gel flash chromatography (0 to 15% EtOAc in DCM) to give1-(2-(2,6-diethylphenyl)-3-(7-fluoro-1H-indol-4-yl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl)-3,3,3-trifluoro-2,2-dimethylpropan-1-one.MS: (ES) m/z calculated for C₃₀H₃₃F₄N₄O [M+H]⁺ 541.3, found 541.2.

Step b: To a solution of1-(2-(2,6-diethylphenyl)-3-(7-fluoro-1H-indol-4-yl)-6,6-dimethyl-2,6-dihydropyrrolo[3,4-c]pyrazol-5(4H)-yl)-3,3,3-trifluoro-2,2-dimethylpropan-1-one(35 mg, 0.064 mmol) in THF (2 mL) was added a solution of DIBAL-H in DCM(1.0 M, 1.5 mL, 1.5 mmol). After 1 h at room temperature, the reactionmixture was quenched with aqueous NaHCO₃, and partitioned between EtOAcand aqueous NaHCO₃. The organic layer was separated, dried over Na₂SO₄,and concentrated in vacuo. The residue was purified by silica gel flashchromatography (0 to 10% EtOAc in DCM) to give4-(2-(2,6-diethylphenyl)-6,6-dimethyl-5-(3,3,3-trifluoro-2,2-dimethylpropyl)-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-7-fluoro-1H-indole.¹H NMR (400 MHz, CDCl₃) δ 8.49 (s, 1H), 7.27 (dd, J=2.4, 2.4 Hz, 1H),7.23 (dd, J=8.0, 8.0 Hz, 1H), 7.06 (d, J=7.2 Hz, 2H), 6.63 (m, 1H), 6.56(m, 1H), 6.47 (m, 1H), 3.93 (s, 2H), 2.84 (s, 2H), 2.35 (m, 2H), 2.20(m, 2H), 1.41 (s, 6H), 1.19 (s, 6H), 0.98 (t, J=7.6 Hz, 6H). MS: (ES)m/z calculated for C₃₀H₃₅F₄N₄ [M+H]⁺ 527.3, found 527.2.

Example 10 Synthesis of4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-3-fluoro-1H-indole

Step a: To a suspension of 4-bromo-3-fluoro-1H-indole (240 mg, 1.1mmol), bis(pinacolato)diboron (420 mg, 1.7 mmol) and KOAc (320 mg, 3.3mmol) in dioxane (5 mL) was added Pd(dppf)Cl₂ complex withdichloromethane (140 mg, 0.17 mmol). The reaction mixture was degassedwith N₂ for 2 min and stirred at 90° C. for 2 h. The reaction mixturewas adsorbed onto silica gel and purified by was purified by silica gelflash chromatography (0 to 100% EtOAc in hexanes) to give3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. MS:(ES) m/z calculated for C₁₄H₁₈BFNO₂ [M+H]⁺ 262.1, found 261.2.

Step b: To a suspension of5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole(74 mg, 0.12 mmol),3-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (64mg, 0.25 mmol) and K₂CO₃ (169 mg, 1.2 mmol) in dioxane (6 mL) and water(1 mL) was added Pd(dppf)Cl₂ complex with dichloromethane (49 mg, 0.060mmol). The reaction mixture was degassed with N₂ for 2 min and stirredunder N₂ at 90° C. for 16 h. The reaction mixture was adsorbed ontosilica gel and purified by silica gel flash chromatography (0 to 30%MTBE in hexanes) followed by HPLC (MeCN/H₂O, with 1% TFA), andpreparative TLC (EtOAc in toluene) to give4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-3-fluoro-1H-indole.¹H NMR (400 MHz, CD₃OD) δ 8.25 (d, J=8.4 Hz, 1H), 7.89-7.94 (m, 2H),7.28 (t, J=7.6 Hz, 1H), 7.20 (dd, J=8.0, 2.6 Hz, 1H), 7.09-7.15 (m, 3H),6.85 (t, J=8.4 Hz, 1H), 6.51 (d, J=7.2 Hz, 1H), 4.19 (s, 2H), 3.65 (s,2H), 2.19-2.50 (m, 4H), 1.54 (s, 6H), 0.87-1.21 (m, 6H). MS: (ES) m/zcalculated for C₃₄H₃₂F₇N₄ [M+H]⁺ 629.3, found 629.3.

Example 11 Synthesis of4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-3-methyl-1H-indole

Step a: To a suspension of 4-bromo-3-methyl-1H-indole (240 mg, 1.1mmol), bis(pinacolato)diboron (910 mg, 3.6 mmol) and KOAc (710 mg, 7.2mmol) in dioxane (6 mL) was added Pd(dppf)Cl₂ complex withdichloromethane (290 mg, 0.36 mmol). The reaction mixture was degassed(N₂) for 2 min and stirred at 90° C. for 4 h. The reaction mixture wasadsorbed onto silica gel and purified by silica gel flash chromatography(0 to 25% MTBE in hexanes) to give3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole. MS:(ES) m/z calculated for C₁₅H₂₁BNO₂ [M+H]⁺ 258.2, found 258.1.

Step b: To a suspension of5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-3-iodo-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazole(65 mg, 0.10 mmol),3-methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (127mg, 0.49 mmol) and K₂CO₃ (256 mg, 1.9 mmol) in dioxane (6 mL) and water(1 mL) was added Pd(dppf)Cl₂ complex with dichloromethane (51 mg, 0.062mmol). The reaction mixture was degassed with N₂ for 2 min and stirredunder N₂ at 90° C. for 2 h. The reaction mixture was adsorbed ontosilica gel and purified by silica gel flash chromatography (0 to 100%MTBE in hexanes) followed by HPLC (MeCN/H₂O, with 1% TFA) andpreparative TLC (40% acetone in hexanes) to give4-(5-(2,4-bis(trifluoromethyl)benzyl)-2-(2,6-diethylphenyl)-6,6-dimethyl-2,4,5,6-tetrahydropyrrolo[3,4-c]pyrazol-3-yl)-3-methyl-1H-indole.¹H NMR (400 MHz, CD₃OD) δ 8.25-8.18 (m, 1H), 7.92 (d, J=9.0 Hz, 2H),7.29-7.17 (m, 3H), 7.04 (d, J=1.2 Hz, 1H), 6.97-6.90 (m, 1H), 6.77 (dd,J=8.2, 7.3 Hz, 1H), 6.44 (dd, J=7.4, 0.9 Hz, 1H), 4.25-4.10 (m, 2H),3.66 (d, J=11.6 Hz, 1H), 3.51 (d, J=11.6 Hz, 1H), 2.28-2.58 (m, 3H),2.17 (s, 3H), 2.00-2.11 (m, 1H), 1.55 (s, 3H), 1.53 (s, 3H), 1.34 (t,J=7.6 Hz, 3H), 0.77 (t, J=7.6 Hz, 3H). MS: (ES) m/z calculated forC₃₅H₃₅F₆N₄ [M+H]⁺ 625.3, found 625.3.

Example 12 Synthesis of tert-butyl2-(2,6-dimethylphenyl)-3-(1H-indol-5-yl)-4,6-dihydropyrrolo[3,4-c]pyrazole-5-carboxylate

Step a: To tert-butyl 3-cyano-4-oxo-pyrrolidine-1-carboxylate (19.5 g,92.54 mmol) and (2,6-dimethylphenyl)hydrazine hydrochloride (16 g, 92.65mmol) were added EtOH (160 mL) and AcOH (40 mL). The resultingsuspension was stirred at 50° C. overnight. Upon completion, thereaction mixture was quenched with 1N NaOH aqueous solution andextracted with EtOAc (2×100 mL), dried (MgSO₄), and concentrated invacuo. The crude product was then purified by silica gel flashchromatography (50% EtOAc in hexanes) to obtain tert-butyl3-amino-2-(2,6-dimethylphenyl)-4,6-dihydropyrrolo[3,4-c]pyrazole-5-carboxylate.MS: (ES) m/z calculated for C₁₈H₂₅N₄O₂ [M+H]⁺ 329.2, found 329.2.

Step b: Isoamylnitrite (11.74 mL, 87.5 mmol) was added slowly at roomtemperature to a mixture of tert-butyl3-amino-2-(2,6-dimethylphenyl)-4,6-dihydropyrrolo[3,4-c]pyrazole-5-carboxylate(14.35 g, 43.75 mmol), diiodomethane (14 mL, 175 mmol) and MeCN (180mL). The resulting reaction mixture was stirred at room temperature for2 h. The reaction mixture was adsorbed onto silica gel and purified bysilica gel flash chromatography (30% EtOAc in hexanes) to obtaintert-butyl2-(2,6-dimethylphenyl)-3-iodo-4,6-dihydropyrrolo[3,4-c]pyrazole-5-carboxylate.MS: (ES) m/z calculated for C₁₈H₂₃IN₃O₂[M+H]⁺ 440.1, found 440.2.

Step c: To a suspension of tert-butyl2-(2,6-dimethylphenyl)-3-iodo-4,6-dihydropyrrolo[3,4-c]pyrazole-5-carboxylate(878 mg, 2 mmol),5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (729 mg, 3mmol) and Na₂CO₃ (530 g, 5 mmol) in dioxane (8 mL) and water (2 mL) wasadded Pd(dppf)Cl₂ complex with dichloromethane (163 mg, 0.2 mmol). Thereaction mixture was degassed with N₂ for 2 min and stirred at 90° C.for 2 h. The reaction mixture was diluted with EtOAc and filteredthrough Celite. The solvent was removed under reduced pressure and theresidue was purified by silica gel flash chromatography (0 to 100% EtOAcin hexanes) to give tert-butyl2-(2,6-dimethylphenyl)-3-(1H-indol-5-yl)-4,6-dihydropyrrolo[3,4-c]pyrazole-5-carboxylate.MS: (ES) m/z calculated for C₂₆H₂₉N₄O₂ [M+H]⁺ 429.2, found 429.2.

Example 13

The compounds in Table 1 and Table 2, below, were prepared using themethods described above. Characterization data (MS and/or NMR) isprovided for each compound listed.

TABLE 1 Structure & NMR/MS Characterization Data of Specific EmbodimentsStructure ¹H NMR MS

¹H NMR (400 MHz, CD₃OD) δ 7.44 (d, J = 3.1 Hz, 1H), 7.32 (t, J = 8.0 Hz,1H), 7.16 (d, J = 7.6 Hz, 2H), 6.59- 6.65 (m, 1H), 6.53-6.56 (m, 1H),6.45-6.50 (m, 1H), 4.72 (s, 2H), 3.88 (s, 2H), 2.19-2.37 (m, 4H), 1.87(s, 6H), 1.46 (s, 9H), 0.99 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculatedfor C₃₂H₃₉FN₅O₃ [M + H]⁺ 560.3, found 560.3.

¹H NMR (400 MHz, CD₃OD) δ 7.89 (br s, 1H), 7.37 (d, J = 3.2 Hz, 1H),7.31 (t, J = 8.0 Hz, 1H), 7.12-7.15 (m, 3H), 6.54-6.60 (m, 1H),6.50-6.53 (m, 1H), 6.39-6.43 (m, 1H), 4.12 (s, 2H), 3.90 (s, 2H),2.15-2.37 (m, 4H), 1.50 (s, 6H), 0.97 (t, J = 7.6 Hz, 6H). MS: (ES) m/zcalculated for C₂₉H₃₁FN₅O [M + H]⁺ 484.3, found 484.3.

¹H NMR (400 MHz, CDCl₃) δ 8.53 (s, 1H), 7.27 (dd, J = 2.6, 2.6 Hz, 1H),7.23 (dd, J = 7.6, 7.6 Hz, 1H), 7.06 (d, J = 7.6, 2H), 6.63 (m, 1H),6.56 (m, 1H), 6.48 (m, 1H), 3.99 (s, 2H), 2.70 (s, 2H), 2.39 (m, 2H),2.23 (m, 2H), 1.62 (br s, 1H), 1.43 (s, 6H), 1.25 (s, 6H), 0.99 (t, J =7.6 Hz, 6H). MS: (ES) m/z calculated for C₂₉H₃₆FN₄O [M + H]⁺ 475.3 found475.3.

¹H NMR (400 MHz, CD₃OD) δ 8.20- 8.27 (m, 1H), 7.89-7.92 (m, 2H), 7.38(s, 1H), 7.22-7.32 (m, 2H), 6.95-6.98 (m, 1H), 6.66 (dd, J = 8.2, 10.8Hz, 1H), 6.50 (dd, J = 4.5, 8.2 Hz, 1H), 4.88 (br, 1H), 4.18 (s, 2H),3.84 (d, J = 11.4 Hz, 1H), 3.49 (d, J = 11.4 Hz, 1H), 2.47 (dq, J = 7.5,15 Hz, 1H), 2.37-2.46 (m, 2H), 2.07 (dq, J = 7.5, 15 Hz, 1H), 1.54 (s,3H), 1.52 (s, 3H), 1.33 (t, J = 7.6 Hz, 3H), 0.76 (t, J = 7.6 Hz, 3H).MS: (ES) m/z calculated for C₃₄H₃₁ClF₇N₄ [M + H]⁺ 663.2, found 663.2.

¹H NMR (400 MHz, CD₃OD) δ 7.56- 7.61 (m, 1H), 7.34 (d, J = 3.0 Hz, 1H),7.30 (t, J = 7.6 Hz, 1H), 7.18-7.22 (m, 1H), 7.14 (d, J = 8.0 Hz, 2H),7.01- 7.07 (m, 1H), 6.53-6.58 (m, 1H), 6.45 (t, J = 3.1 Hz, 1H),6.38-6.42 (m, 1H), 4.04 (s, 2H), 3.73 (s, 2H), 2.15-2.38 (m, 4H), 1.57(s, 6H), 0.98 (t, J = 7.2 Hz, 6H). MS: (ES) m/z calculated forC₃₂H₃₂ClF₂N₄ [M + H]⁺ 545.2, found 545.2.

¹H NMR (400 MHz, CD₃OD) δ 7.50 (m, 1H), 7.37-7.26 (m, 2H), 7.14 (d, J =7.7 Hz, 2H), 6.91 (t, J = 8.7 Hz, 2H), 6.55 (dd, J = 10.9, 8.2 Hz, 1H),6.48-6.36 (m, 2H), 3.96 (s, 2H), 3.73 (s, 2H), 2.26 (m, 4H), 1.56 (s,6H), 0.97 (t, J = 7.6, 6H). MS: (ES) m/z calculated for C₃₂H₃₂F₃N₄ [M +H]⁺ 529.3, found 529.3.

¹H NMR TFA salt (400 MHz, CD₃OD) δ 11.41 (s, 1H), 8.75 (d, J = 5.6 Hz,2H), 7.94 (d, J = 6.4 Hz, 2H), 7.39 (dd, J = 2.6, 2.6 Hz, 1H), 7.34 (dd,J = 7.6, 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 2H), 6.60 (m, 1H), 6.45 (m,2H), 4.67 (s, 2H), 4.39 (s, 2H), 2.25 (m, 4H), 1.88 (s, 6H), 0.98 (t, J= 7.6 Hz, 6H). MS: (ES) m/z calculated for C₃₁H₃₃FN₅ [M + H]⁺ 494.3,found 494.3.

¹H NMR (400 MHz, CD₃OD) δ 7.47- 7.57 (m, 1H), 7.39 (s, 1H), 7.23-7.28(m, 2H), 7.12-7.16 (m, 2H), 6.95- 6.98 (m, 1H), 6.66 (dd, J = 8.2, 10.8Hz, 1H), 6.50 (dd, J = 4.5, 8.2 Hz, 1H), 4.88 (br, 1H), 4.00 (d, J =11.4 Hz, 1H), 3.81-3.90 (m, 2H), 3.50 (d, J = 11.4 Hz, 1H), 2.33-2.46(m, 3H), 2.00-2.13 (m, 1H), 1.56 (s, 3H), 1.49 (s, 3H), 1.31 (t, J = 7.6Hz, 3H), 0.75 (t, J = 7.6 Hz, 3H). MS: (ES) m/z calculated forC₃₂H₃₁Cl₂F₂N₄ [M + H]⁺ 579.2, found 579.2.

¹H NMR TFA salt (400 MHz, CD₃OD) δ 11.46 (s, 1H), 8.87 (d, J = 1.0 Hz,1H), 8.71 (d, J = 5.2 Hz, 1H), 8.26 (d, J = 7.6 Hz, 1H), 7.68 (m, 1H),7.41 (dd, J = 2.8, 2.8 Hz, 1H), 7.35 (dd, J = 7.8, 7.8 Hz, 1H), 7.18 (d,J = 8.0 Hz, 2H), 6.61 (m, 1H), 6.45 (m, 2H), 4.75 (s, 2H), 4.53 (s, 2H),2.25 (m, 4H), 1.97 (s, 6H), 0.97 (t, J = 7.6 Hz, 6H). MS: (ES) m/zcalculated for C₃₁H₃₃FN₅ [M + H]⁺ 494.3, found 494.3.

¹H NMR (400 MHz, CD₃OD) δ 7.46- 7.52 (m, 1H), 7.39 (s, 1H), 7.23-7.28(m, 2H), 6.82-7.00 (m, 3H), 6.66 (dd, J = 8.2, 10.8 Hz, 1H), 6.50 (dd, J= 4.5, 8.2 Hz, 1H), 4.88 (br, 1H), 4.00 (d, J = 11.6 Hz, 1H), 3.81-3.90(m, 2H), 3.50 (d, J = 11.6 Hz, 1H), 2.30-2.58 (m, 3H), 2.00-2.12 (m,1H), 1.57 (s, 3H), 1.49 (s, 3H), 1.31 (t, J = 7.6 Hz, 3H), 0.75 (t, J =7.6 Hz, 3H). MS: (ES) m/z calculated for C₃₂H₃₁ClF₃N₄ [M + H]⁺ 563.2,found 563.2.

¹H NMR (400 MHz, CD₃OD) δ 7.26- 7.34 (m, 3H), 7.16-7.25 (m, 2H), 7.13(d, J = 7.2 Hz, 2H), 6.90-7.00 (m, 1H), 6.55 (dd, J = 10.9, 8.1 Hz, 1H),6.36-6.46 (m, 2H), 5.46-5.51 (m, 1H), 3.93 (s, 2H), 3.67 (s, 2H), 2.05-2.45 (m, 4H), 1.54 (s, 6H), 0.98 (t, J = 7.6 Hz, 6H). MS: (ES) m/zcalculated for C₃₂H₃₃F₂N₄ [M + H]⁺ 511.3, found 511.3.

¹H NMR TFA salt (400 MHz, CD₃OD) δ 11.46 (s, 1H), 8.68 (d, J = 4.8 Hz,1H), 7.92 (dd, J = 1.6, 7.6 Hz, 1H), 7.62 (d, J = 8.0, 1H), 7.45 (m,2H), 7.38 (dd, J = 7.6, 7.6 Hz, 1H), 7.18 (d, J = 7.6 Hz, 2H), 6.62 (m,1H), 6.53 (m, 1H), 6.45 (m, 1H), 4.79 (s, 2H), 4.64 (s, 2H), 2.27 (m,4H), 1.92 (s, 6H), 0.98 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculated forC₃₁H₃₃FN₅ [M + H]⁺ 494.3, found 494.3.

¹H NMR (400 MHz, CD₃OD) δ 7.57 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 2.1 Hz,1H), 7.26-7.37 (m, 3H), 7.14 (d, J = 7.7 Hz, 2H), 6.66 (dd, J = 8.2,10.8 Hz, 1H), 6.39-6.46 (m, 2H), 4.88 (br, 1H), 4.04 (s, 2H), 3.74 (s,2H), 2.03-2.33 (m, 4H), 1.56 (s, 6H), 0.98 (t, J = 7.6 Hz, 6H). MS: (ES)m/z calculated for C₃₂H₃₂Cl₂FN₄ [M + H]⁺ 561.2, found 561.2.

¹H NMR (400 MHz, CD₃OD) δ 7.42 (d, J = 8.4 Hz, 2H), 7.28-7.34 (m, 4H),7.20-7.26 (m, 1H), 7.14 (d, J = 7.7 Hz, 2H), 6.55 (dd, J = 8.2, 11.0 Hz,1H), 6.47-6.35 (m, 2H), 3.93 (s, 2H), 3.68 (s, 2H), 2.12-2.39 (m, 4H),1.56 (s, 6H), 0.97 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculated forC₃₂H₃₄FN₄ [M + H]⁺ 493.3, found 493.3.

¹H NMR (400 MHz, CD₃OD) δ 7.32- 7.49 (m, 1H), 7.25-7.34 (m, 3H),7.04-7.14 (m, 4H), 6.53-6.57 (m, 1H), 6.38-6.46 (m, 2H), 4.88 (br, 1H),3.99 (s, 2H), 3.74 (s, 2H), 2.17-2.36 (m, 4H), 1.57 (s, 6H), 0.97 (t, J= 7.6 Hz, 6H). MS: (ES) m/z calculated for C₃₂H₃₃F₂N₄ [M + H]⁺ 511.3,found 511.3.

¹H NMR (400 MHz, CD₃OD) δ 7.39- 7.45 (m, 2H), 7.26-7.36 (m, 2H), 7.14(d, J = 8.2 Hz, 2H), 6.98-7.04 (m, 2H), 6.55 (dd, J = 8.2, 11.0 Hz, 1H),6.35- 6.46 (m, 2H), 4.88 (br, 1H), 3.91 (s, 2H), 3.66 (s, 2H), 2.17-2.36(m, 4H), 1.55 (s, 6H), 0.97 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculatedfor C₃₂H₃₃F₂N₄ [M + H]⁺ 511.3, found 511.3.

¹H NMR (400 MHz, CD₃OD) δ 8.23 (d, J = 8.2 Hz, 1H), 7.92-7.93 (m, 2H),7.27-7.39 (m. 2H), 7.15 (d, J = 7.7 Hz, 2H), 6.83 (d, J = 7.7 Hz, 1H),6.42- 6.48 (m, 2H), 4.88 (br, 1H), 4.22 (s, 2H), 3.66 (s, 2H), 2.20-2.36(m, 4H), 1.56 (s, 6H), 1.00 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculatedfor C₃₄H₃₂ClF₆N₄ [M + H]⁺ 645.2, found 645.2.

¹H NMR (400 MHz, CD₃OD) δ 8.24 (d, J = 8.6 Hz, 1H), 7.89-7.95 (m, 2H),7.24-7.34 (m, 2H), 7.13 (d, J = 7.7 Hz, 2H), 6.60 (d, J = 7.2 Hz, 1H),6.33- 6.42 (m, 2H), 4.22 (s, 2H), 3.73 (s, 2H), 2.16-2.42 (m, 7H), 1.56(s, 6H), 0.99 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculated forC₃₅H₃₅F₆N₄ [M + H]⁺ 625.3, found 625.3.

¹H NMR (400 MHz, CD₃OD) δ 8.25- 8.29 (m, 1H), 7.95-7.97 (m, 2H),7.21-7.29 (m, 4H), 7.07 (d, J = 1.4 Hz, 1H), 6.47 (dd, J = 1.4, 12.6 Hz,1H), 6.33-6.35 (m, 1H), 4.88 (br, 1H), 4.25 (s, 2H), 3.93 (s, 2H),2.26-2.35 (m, 4H), 1.53 (s, 6H), 1.04 (t, J = 7.6 Hz, 6H). MS: (ES) m/zcalculated for C₃₄H₃₂F₇N₄ [M + H]⁺ 629.2, found 629.3.

¹H NMR TFA salt (400 MHz, CD₃OD) δ 11.31 (s, 1H), 8.56 (d, J = 4.4 Hz,1H), 7.99 (dd, J = 1.6, 7.2 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H), 7.50 (m,1H), 7.32 (m, 2H), 7.15 (d, J = 7.6 Hz, 2H), 6.58 (m, 1H), 6.44 (m, 1H),6.39 (m, 1H), 4.71 (s, 2H), 2.28 (m, 4H), 2.03 (s, 6H), 0.99 (t, J = 7.6Hz, 6H). MS: (ES) m/z calculated for C₃₁H₃₁FN₅O [M + H]⁺ 508.2, found508.2.

¹H NMR (400 MHz, CDCl₃) δ 8.30 (s, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.51(d, J = 8.0 Hz, 1H), 7.40 (dd, J = 7.6, 7.6 Hz, 1H), 7.14 (m, 3H), 6.97(d, J = 7.6 Hz, 2H), 6.51 (m, 1H), 6.41 (m, 1H), 6.36 (m, 1H), 4.00 (s,2H), 3.62 (s, 2H), 2.28 (m, 2H), 2.12 (m, 2H), 1.47 (s, 6H), 0.90 (t, J= 7.4 Hz, 6H). MS: (ES) m/z calculated for C₃₃H₃₃F₄N₄ [M + H]⁺ 561.3,found 561.3.

¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 7.26 (dd, J = 2.8, 2.8 Hz, 1H),7.16 (dd, J = 7.6, 7.6 Hz, 1H), 6.99 (d, J = 7.6 Hz, 2H), 6.59 (m, 1H),6.43 (m, 2H), 4.53 (s, 2H), 2.20 (m, 6H), 1.84 (s, 6H), 0.91 (m, 13H).MS: (ES) m/z calculated for C₃₀H₃₆FN₄O [M + H]⁺ 487.3, found 487.3.

¹H NMR (400 MHz, CDCl₃) δ 8.19 (s, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.78(s, 1H), 7.67 (d, J = 8.4 Hz, 1H), 7.17 (dd, J = 7.8, 7.8 Hz, 1H), 7.09(dd, J = 2.8, 2.8 Hz, 1H), 7.01 (d, J = 7.6 Hz, 2H), 6.81 (dd, J = 2.0,8.8 Hz, 1H), 6.35 (m, 1H), 6.25 (dd, J = 2.0, 10.8 Hz, 1H), 4.06 (s,2H), 3.64 (s, 2H), 2.28 (m, 2H), 2.15 (m, 2H), 1.48 (s, 6H), 0.92 (t, J= 7.6 Hz, 6H). MS: (ES) m/z calculated for C₃₄H₃₂F₇N₄ [M + H]⁺ 629.2,found 629.2.

¹H NMR (400 MHz, CDCl₃) δ 8.54 (s, 1H), 7.34 (dd, J = 2.8, 2.8 Hz, 1H),7.26 (m, 1H), 7.07 (d, J = 7.6 Hz, 2H), 6.68 (m, 1H), 6.49 (m, 2H), 4.85(s, 2H), 2.84 (s, 2H), 2.32 (m, 2H), 2.20 (m, 2H), 1.91 (s, 6H), 1.54(s, 6H), 1.00 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculated forC₃₀H₃₃F₄N₄O [M + H]⁺ 541.3, found 541.2.

¹H NMR (400 MHz, CD₃OD) δ 8.21 (d, J = 8.4 Hz, 1H), 7.90 (br s, 2H),7.24- 7.31 (m, 4H), 6.92-6.95 (m, 1H), 6.88 (t, J = 8.4 Hz), 6.55 (dd, J= 0.76, 7.6 Hz, 1H), 4.18 (s, 2H), 3.86 (d, J = 12 Hz, 1H), 3.48 (d, J =11.6 Hz, 1H), 2.03-2.61 (m, 4H), 1.54 (s, 3H), 1.53 (s, 3H), 1.35 (t, J= 7.6 Hz, 3H), 0.75 (t, J = 7.6 Hz, 3H). MS: (ES) m/z calculated forC₃₄H₃₂ClF₆N₄ [M + H]⁺ 645.2, found 645.2.

¹H NMR (400 MHz, CD₃OD) δ 8.38 (d, J = 8.7 Hz, 1H), 8.06 (s, 1H), 7.88(d, J = 4.5 Hz, 2H), 7.41 (dd, J = 8.3, 0.8 Hz, 1H), 7.23-7.33 (m, 2H),6.93- 7.08 (m, 2H), 6.68-6.75 (m, 1H), 4.21 (s, 2H), 3.99 (d, J = 11.3Hz, 1H), 3.51 (d, J = 11.4 Hz, 1H), 2.46-2.68 (m, 2H), 2.30-2.40 (m,1H), 1.98-2.06 (m, 1H), 1.57 (s, 3H), 1.55 (s, 3H), 1.36 (t, J = 7.5 Hz,3H), 0.77 (t, J = 7.6 Hz, 3H). MS: (ES) m/z calculated for C₃₅H₃₂F₆N₅[M + H]⁺ 636.3, found 636.3.

¹H NMR (400 MHz, CDCl₃) δ 8.42 (s, 1H), 8.20 (d, J = 8.4 Hz, 1H), 7.86(s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.21 (m, 2H), 7.03 (br s, 2H), 6.51(t, J = 10.4 Hz, 1H), 6.38 (m, 1H), 4.14 (s, 2H), 3.63 (s, 2H), 2.38 (brs, 4H), 1.56 (s, 6H), 1.00 (br s, 6H). MS: (ES) m/z calculated forC₃₄H₃₁F₈N₄ [M + H]⁺ 647.2, found 647.2.

¹H NMR (400 MHz, CD₃OD) δ 8.34 (s, 1H), 7.89 (d, J = 8.2 Hz, 1H), 7.69-7.77 (m, 1H), 7.22-7.35 (m, 3H), 7.14 (d, J = 7.7 Hz, 2H), 6.80 (t, J =7.8 Hz, 1H), 6.49 (m, 1H), 6.34 (m, 1H), 4.22 (s, 2H), 3.71 (s, 2H),2.20-2.50 (m, 4H), 1.56 (s, 6H), 0.99 (m, 6H). MS: (ES) m/z calculatedfor C₃₄H₃₃F₆N₄ [M + H]⁺ 611.3, found 611.3.

¹H NMR (400 MHz, CD₃OD) δ 7.38 (d, J = 3.0 Hz, 1H), 7.24-7.35 (m, 4H),7.15 (d, J = 7.6 Hz, 2H), 6.90-7.01 (m, 3H), 6.85 (t, J = 7.6 Hz, 1H),6.54 (d, J = 7.2 Hz, 1H), 6.48 (d, J = 3.1 Hz, 1H), 4.82 (s, 2H),2.18-2.42 (m, 4H), 1.91 (s, 6H), 0.99 (t, J = 7.6 Hz, 6H). MS: (ES) m/zcalculated for C₃₃H₃₅N₄O₂ [M + H]⁺ 519.3, found 519.3.

¹H NMR (400 MHz, CD₃OD) δ 7.63 (d, J = 2.7 Hz, 1H), 7.20-7.40 (m, 5H),7.13 (d, J = 7.7 Hz, 2H), 6.76-6.85 (m, 1H), 6.49 (dt, J = 7.5, 0.7 Hz,1H), 6.40 (dd, J = 3.2, 0.8 Hz, 1H), 4.06 (s, 2H), 3.76 (s, 2H), 3.31(s, 2H), 2.15- 2.45 (m, 4H), 1.57 (s, 6H), 0.94 (t, J = 7.6 Hz, 6H). MS:(ES) m/z calculated for C₃₂H₃₃Cl₂N₄ [M + H]⁺ 543.2, found 543.2.

¹H NMR (400 MHz, CD₃OD) δ 8.23 (d, J = 8.2 Hz, 1H), 7.92-7.93 (m, 2H),7.22-7.39 (m, 3H), 7.07 (br, 2H), 6.65 (dd, J = 8.2, 10.8, 1H),6.23-6.27 (m, 1H), 4.87 (br, 1H), 4.21 (s, 2H), 3.67 (s, 2H), 2.20-2.36(br, 4H), 1.57 (s, 6H), 0.88-1.02 (br, 6H). MS: (ES) m/z calculated forC₃₄H₃₂F₇N₄ [M + H]⁺ 629.2, found 629.3.

¹H NMR (400 MHz, CD₃OD) δ 8.24 (d, J = 7.9 Hz, 1H), 7.54-7.64 (m, 2H),7.23-7.42 (m, 4H), 7.13 (d, J = 7.7 Hz, 2H), 6.80 (t, J = 7.7 Hz, 1H),6.35- 6.49 (m, 2H), 4.86 (br, 1H), 4.13 (s, 2H), 3.70 (s, 2H), 2.17-2.42(m, 4H), 1.55 (s, 6H), 0.99 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculatedfor C₃₃H₃₄F₃N₄ [M + H]⁺ 543.3, found 543.6.

¹H NMR (400 MHz, CD₃OD) δ 8.24 (d, J = 8.2 Hz, 1H), 7.90-7.93 (m, 2H),7.26-7.32 (m, 3H), 7.14 (d, J = 7.7 Hz, 2H), 6.80 (t, J = 7.7 Hz, 1H),6.35- 6.49 (m, 2H), 4.88 (br, 1H), 4.22 (s, 2H), 3.74 (s, 2H), 2.20-2.36(m, 4H), 1.56 (s, 6H), 0.99 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculatedfor C₃₄H₃₃F₆N₄ [M + H]⁺ 611.3, found 611.6.

¹H NMR (400 MHz, CD₃OD) δ 7.68 (s, 1H), 7.37-7.46 (m, 2H), 7.14-7.32 (m,5H), 7.07 (d, J = 1.4 Hz, 1H), 6.84-6.86 (m, Hz, 1H), 6.25-6.26 (m, 1H),4.86 (br, 1H), 4.08 (s, 2H), 3.94 (s, 2H), 2.26-2.35 (m, 4H), 1.53 (s,6H), 1.03 (t, J = 7.6 Hz, 6H). MS: (ES) m/z calculated for C₃₂H₃₃Cl₂N₄[M + H]⁺ 543.2, found 543.5.

¹H NMR (400 MHz, CD₃OD) δ 8.01 (d, J = 8.0 Hz, 1H), 7.58-7.73 (m, 2H),7.42 (t, J = 7.7 Hz, 2H), 7.13-7.28 (m, 5H), 6.82 (dd, J = 1.7, 8.0 Hz,1H), 6.23-6.24 (m, 1H), 4.88 (br, 1H), 4.16 (s, 2H), 3.88 (s, 2H),2.20-2.36 (m, 4H), 1.52 (s, 6H), 1.03 (t, J = 7.6 Hz, 6H). MS: (ES) m/zcalculated for C₃₃H₃₄F₃N₄ [M + H]⁺ 543.3, found 543.5.

¹H NMR (400 MHz, CDCl₃) δ 8.13 (br, 1H), 7.32-7.37 (m, 1H), 7.15- 7.22(m, 4H), 7.06 (d, J = 7.6 Hz, 1H), 6.91 (dd, J = 1.7, 8.5 Hz, 1H), 6.43-6.44 (m, 1H), 4.08 (s, 2H), 2.50 (s, 2H), 2.00 (s, 6H), 1.36 (s, 6H),1.00 (s, 9H). MS: (ES) m/z calculated for C₂₈H₃₅N₄ [M + H]⁺ 427.3, found427.5.

¹H NMR (400 MHz, CD₃OD) δ 7.55- 7.63 (m, 2H), 7.10-7.39 (m, 9H),6.82-6.90 (m, 1H), 6.23-6.24 (m, 1H), 4.82 (br, 2H), 4.64 (d, J = 11.5Hz, 1H), 4.33 (d, J = 11.5 Hz, 1H), 3.87 (s, 1H), 2.06-2.39 (m, 4H),1.59 (s, 3H), 1.35 (s, 3H), 1.19 (s, 3H), 1.05 (s, 3H), 0.83-1.03 (m,6H). MS: (ES) m/z calculated for C₃₅H₄₁N₄O [M + H]⁺ 533.3, found 533.7.

¹H NMR (400 MHz, CDCl₃) δ 8.15 (br, 1H), 7.48-7.56 (m, 2H), 7.24-7.44(m, 5H), 7.08-7.20 (m, 4H), 6.83 (dd, J = 1.7, 8.6 Hz, 1H), 6.36-6.37(m, 1H), 4.93 (s, 1H), 4.69 (d, J = 11.4 Hz, 1H), 4.18-4.35 (m, 4H),3.94 (d, J = 11.4 Hz, 1H), 2.14-2.43 (m, 4H), 1.58 (s, 3H), 1.51 (s,3H), 1.19-1.34 (m, 6H), 0.96-1.07 (m, 6H). MS: (ES) m/z calculated forC₃₅H₃₉N₄O₂ [M + H]⁺ 547.3, found 547.5.

¹H NMR (400 MHz, CDCl₃) δ δ 8.17 (br, 1H), 7.13-7.37 (m, 4H), 7.03- 7.06(m, 2H), 6.89 (dd, J = 1.1, 8.5 Hz, 1H), 6.42-6.44 (m, 1H), 4.18 (s,2H), 4.06 (s, 2H), 2.69 (s, 2H), 2.00 (s, 6H), 1.01 (s, 9H). MS: (ES)m/z calculated for C₂₆H₃₁N₄ [M + H]⁺ 399.3, found 399.5.

TABLE 2 Structure & MS Characterization Data of Specific EmbodimentsStructure MS

MS: (ES) m/z calculated for C₂₉H₃₅FN₅O [M + H]⁺ 488.3, found 488.3.

MS: (ES) m/z calculated for C₃₅H₃₂F₆N₄O₂ [M + H]⁺ 655.2, found 655.2.

MS: (ES) m/z calculated for C₂₇H₃₁FN₅O [M + H]⁺ 460.3, found 460.3.

MS: (ES) m/z calculated for C₃₄H₃₃F₆N₄O [M + H]⁺ 627.3, found 627.3.

MS: (ES) m/z calculated for C₃₂H₃₄F₃N₆O [M + H]⁺ 575.3, found 575.3.

MS: (ES) m/z calculated for C₃₄H₃₃F₆N₄O [M + H]⁺ 627.3, found 627.3.

MS: (ES) m/z calculated for C₃₀H₃₇N₄O₂ [M + H]⁺ 485.3, found 485.5.

MS: (ES) m/z calculated for C₂₅H₃₀N₅ [M + H]⁺ 400.2, found 400.5.

MS: (ES) m/z calculated for C₂₇H₃₁N₄O₂ [M + H]⁺ 443.2, found 443.2.

MS: (ES) m/z calculated for C₂₆H₂₇N₄O₃ [M + H]⁺ 443.2, found 443.2.

MS: (ES) m/z calculated for C₂₉H₃₃N₄O [M + H]⁺ 453.2, found 453.2.

MS: (ES) m/z calculated for C₂₈H₁₃N₄O [M + H]⁺ 439.2, found 439.2.

MS: (ES) m/z calculated for C₂₅H₂₈N₅O₂ [M + H]⁺ 430.2, found 430.2.

MS: (ES) m/z calculated for C₂₇H₂₉N₄O [M + H]⁺ 425.2, found 425.2.

MS: (ES) m/z calculated for C₃₀H₃₅N₄O [M + H]⁺ 467.3, found 467.5.

MS: (ES) m/z calculated for C₂₇H₃₁N₄O [M + H]⁺ 427.2, found 427.2.

MS: (ES) m/z calculated for C₂₆H₂₉N₄O₂ [M + H]⁺ 429.2, found 429.5.

MS: (ES) m/z calculated for C₃₁H₃₃N₄O₂S [M + H]⁺ 525.2, found 525.2.

MS: (ES) m/z calculated for C₃₂H₃₃N₄O [M + H]⁺ 489.3, found 489.3.

MS: (ES) m/z calculated for C₂₆H₃₀N₅O [M + H]⁺ 428.2, found 428.2.

MS: (ES) m/z calculated for C₂₈H₂₅N₄O [M + H]⁺ 433.2, found 433.2.

Example 14

This example illustrates the evaluation of the biological activityassociated with specific compounds of the invention.

Materials and Methods

A. Cells

1. C5a Receptor Expressing Cells

a) U937 Cells

U937 cells are a monocytic cell line which express C5aR, and areavailable from ATCC (VA). These cells were cultured as a suspension inRPMI-1640 medium supplemented with 2 mM L-glutamine, 1.5 g/L sodiumbicarbonate, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, and 10%FBS. Cells were grown under 5% CO₂/95% air, 100% humidity at 37° C. andsubcultured twice weekly at 1:6 (cells were cultured at a density rangeof 1×10⁵ to 2×10⁶ cells/mL) and harvested at 1×10⁶ cells/mL. Prior toassay, cells are treated overnight with 0.5 mM of cyclic AMP (Sigma,Ohio) and washed once prior to use. cAMP treated U937 cells can be usedin C5aR ligand binding and functional assays.

b) Isolated Human Neutrophils

Optionally, human or murine neutrophils can be used to assay forcompound activity. Neutrophils may be isolated from fresh human bloodusing density separation and centrifigation. Briefly, whole blood isincubated with equal parts 3% dextran and allowed to separate for 45minutes. After separation, the top layer is layered on top of 15 mls ofFicoll (15 mls of Ficoll for every 30 mls of blood suspension) andcentrifuged for 30 minutes at 400×g with no brake. The pellet at thebottom of the tube is then isolated and resuspended into PharmLyse RBCLysis Buffer (BD Biosciences, San Jose, Calif.) after which the sampleis again centrifuged for 10 minutes at 400×g with brake. The remainingcell pellet is resuspended as appropriate and consists of isolatedneutrophils.

B. Assays

1. Inhibition of C5aR Ligand Binding

cAMP treated U937 cells expressing C5aR were centrifuged and resuspendedin assay buffer (20 mM HEPES pH 7.1, 140 mM NaCl, 1 mM CaCl₂, 5 mMMgCl₂, and with 0.1% bovine serum albumin) to a concentration of 3×10⁶cells/mL. Binding assays were set up as follows. 0.1 mL of cells wasadded to the assay plates containing 5 μL of the compound, giving afinal concentration of ˜2-10 μM each compound for screening (or part ofa dose response for compound IC₅₀ determinations). Then 0.1 mL of ¹²⁵Ilabeled C5a (obtained from Perkin Elmer Life Sciences, Boston, Mass.)diluted in assay buffer to a final concentration of ˜50 pM, yielding˜30,000 cpm per well, was added, the plates sealed and incubated forapproximately 3 hours at 4° C. on a shaker platform. Reactions wereaspirated onto GF/B glass filters pre-soaked in 0.3% polyethyleneimine(PEI) solution, on a vacuum cell harvester (Packard Instruments;Meriden, Conn.). Scintillation fluid (40 μl; Microscint 20, PackardInstruments) was added to each well, the plates were sealed andradioactivity measured in a Topcount scintillation counter (PackardInstruments). Control wells containing either diluent only (for totalcounts) or excess C5a (1 μg/mL, for non-specific binding) were used tocalculate the percent of total inhibition for compound. The computerprogram Prism from GraphPad, Inc. (San Diego, Ca) was used to calculateIC₅₀ values. IC₅₀ values are those concentrations required to reduce thebinding of radiolabeled C5a to the receptor by 50%. (For furtherdescriptions of ligand binding and other functional assays, seeDairaghi, et al., J. Biol. Chem. 274:21569-21574 (1999), Penfold, etal., Proc. Natl. Acad. Sci. USA. 96:9839-9844 (1999), and Dairaghi, etal., J. Biol. Chem. 272:28206-28209 (1997)).

2. Calcium Mobilization

Optionally, compounds may be further assayed for their ability toinhibit calcium flux in cells. To detect the release of intracellularstores of calcium, cells (e.g., cAMP stimulated U937 or neutrophils) areincubated with 3 μM of INDO-1AM dye (Molecular Probes; Eugene, Oreg.) incell media for 45 minutes at room temperature and washed with phosphatebuffered saline (PBS). After INDO-1AM loading, the cells are resuspendedin flux buffer (Hank's balanced salt solution (HBSS) and 1% FBS).Calcium mobilization is measured using a Photon Technology Internationalspectrophotometer (Photon Technology International; New Jersey) withexcitation at 350 nm and dual simultaneous recording of fluorescenceemission at 400 nm and 490 nm. Relative intracellular calcium levels areexpressed as the 400 nm/490 nm emission ratio. Experiments are performedat 37° C. with constant mixing in cuvettes each containing 10⁶ cells in2 mL of flux buffer. The chemokine ligands may be used over a range from1 to 100 nM. The emission ratio is plotted over time (typically 2-3minutes). Candidate ligand blocking compounds (up to 10 μM) are added at10 seconds, followed by chemokines at 60 seconds (i.e., C5a; R&DSystems; Minneapolis, Minn.) and control chemokine (i.e., SDF-1α; R&DSystems; Minneapolis, Minn.) at 150 seconds.

3. Chemotaxis Assays

Optionally, compounds may be further assayed for their ability toinhibit chemotaxis in cells. Chemotaxis assays are performed using 5 μmpore polycarbonate, polyvinylpyrrolidone-coated filters in 96-wellchemotaxis chambers (Neuroprobe; Gaithersburg, Md.) using chemotaxisbuffer (Hank's balanced salt solution (HBSS) and 1% FBS). C5aR ligands(i.e., C5a, R&D Systems; Minneapolis, Minn.) are use to evaluatecompound mediated inhibition of C5aR mediated migration. Otherchemokines (i.e., SDF-1α; R&D Systems; Minneapolis, Minn.) are used asspecificity controls. The lower chamber is loaded with 29 μl ofchemokine (i.e., 0.03 nM C5a) and varying amounts of compound; the topchamber contains 100,000 U937 or neutrophil cells in 20 μl. The chambersare incubated 1.5 hours at 37° C., and the number of cells in the lowerchamber quantified either by direct cell counts in five high poweredfields per well or by the CyQuant assay (Molecular Probes), afluorescent dye method that measures nucleic acid content andmicroscopic observation.

C. Identification of Inhibitors of C5aR

1. Assay

To evaluate small organic molecules that prevent the C5a receptor frombinding ligand, an assay was employed that detected radioactive ligand(i.e, C5a) binding to cells expressing C5aR on the cell surface (forexample, cAMP stimulated U937 cells or isolated human neutrophils). Forcompounds that inhibited binding, whether competitive or not, fewerradioactive counts are observed when compared to uninhibited controls.

Equal numbers of cells were added to each well in the plate. The cellswere then incubated with radiolabeled C5a. Unbound ligand was removed bywashing the cells, and bound ligand was determined by quantifyingradioactive counts. Cells that were incubated without any organiccompound gave total counts; non-specific binding was determined byincubating the cells with unlabeled ligand and labeled ligand. Percentinhibition was determined by the equation:% inhibition=(1−[(sample cpm)−(nonspecific cpm)]/[(totalcpm)−(nonspecific cpm)])×100.

2. Dose Response Curves

To ascertain a candidate compound's affinity for C5aR as well as confirmits ability to inhibit ligand binding, inhibitory activity was titeredover a 1×10⁻¹⁰ to 1×10⁻⁴ M range of compound concentrations. In theassay, the amount of compound was varied; while cell number and ligandconcentration were held constant.

D. In Vivo Efficacy Models

The compounds of interest can be evaluated for potential efficacy intreating a C5a mediated conditions by determining the efficacy of thecompound in an animal model. In addition to the models described below,other suitable animal models for studying the compound of interest canbe found in Mizuno, M. et al., Expert Opin. Investig. Drugs (2005),14(7), 807-821, which is incorporated herein by reference in itsentirety.

1. Models of C5a Induced Leukopenia

a) C5a Induced Leukopenia in a Human C5aR Knock-in Mouse Model

To study the efficacy of compounds of the instant invention in an animalmodel, a recombinant mouse can be created using standard techniques,wherein the genetic sequence coding for the mouse C5aR is replaced withsequence coding for the human C5aR, to create a hC5aR-KI mouse. In thismouse, administration of hC5a leads to upregulation of adhesionmolecules on blood vessel walls which bind blood leukocytes,sequestering them from the blood stream. Animals are administered 20ug/kg of hC5a and 1 minute later leukocytes are quantified in peripheralblood by standard techniques. Pretreatment of mice with varying doses ofthe present compounds can almost completely block the hC5a inducedleukopenia.

b) C5a Induced Leukopenia in a Cynomolgus Model

To study the efficacy of compounds of the instant invention in anon-human primate model model, C5a induced leucopenia is studied in acynomolgus model. In this model administration of hC5a leads toupregulation of adhesion molecules on blood vessel walls which bindblood leukocytes, hence sequestering them from the blood stream. Animalsare administered 10 ug/kg of hC5a and 1 minute later leukocytes arequantified in peripheral blood.

Mouse Model of ANCA Induced Vasculitis

On day 0 hC5aR-KI mice are intraveneously injected with 50 mg/kgpurified antibody to myeloperoxidase (Xiao et al, J. Clin. Invest. 110:955-963 (2002)). Mice are further dosed with oral daily doses ofcompounds of the invention or vehicle for seven days, then mice aresacrificed and kidneys collected for histological examination. Analysisof kidney sections can show significantly reduced number and severity ofcrescentic and necrotic lesions in the glomeruli when compared tovehicle treated animals.

2. Mouse Model of Choroidal Neovascularization

To study the efficacy of compounds of the instant invention in treatmentof age related macular degeneration (AMD) the bruch membrane in the eyesof hC5aR-KI mice are ruptured by laser photocoagulation (Nozika et al,PNAS 103: 2328-2333 (2006). Mice are treated with vehicle or a dailyoral or appropriate intra-vitreal dose of a compound of the inventionfor one to two weeks. Repair of laser induced damage andneovascularization are assessed by histology and angiography.

3. Rheumatoid Arthritis Models

a) Rabbit Model of Destructive Joint Inflammation

To study the effects of candidate compounds on inhibiting theinflammatory response of rabbits to an intra-articular injection of thebacterial membrane component lipopolysaccharide (LPS), a rabbit model ofdestructive joint inflammation is used. This study design mimics thedestructive joint inflammation seen in arthritis. Intra-articularinjection of LPS causes an acute inflammatory response characterized bythe release of cytokines and chemokines, many of which have beenidentified in rheumatoid arthritic joints. Marked increases inleukocytes occur in synovial fluid and in synovium in response toelevation of these chemotactic mediators. Selective antagonists ofchemokine receptors have shown efficacy in this model (see Podolin, etal., J. Immunol. 169(11):6435-6444 (2002)).

A rabbit LPS study is conducted essentially as described in Podolin, etal. ibid., female New Zealand rabbits (approximately 2 kilograms) aretreated intra-articularly in one knee with LPS (10 ng) together witheither vehicle only (phosphate buffered saline with 1% DMSO) or withaddition of candidate compound (dose 1=50 μM or dose 2=100 μM) in atotal volume of 1.0 mL. Sixteen hours after the LPS injection, knees arelavaged and cells counts are performed. Beneficial effects of treatmentwere determined by histopathologic evaluation of synovial inflammation.Inflammation scores are used for the histopathologic evaluation:1—minimal, 2—mild, 3—moderate, 4—moderate-marked.

b) Evaluation of a Compound in a Rat Model of Collagen Induced Arthritis

A 17 day developing type II collagen arthritis study is conducted toevaluate the effects of a candidate compound on arthritis inducedclinical ankle swelling. Rat collagen arthritis is an experimental modelof polyarthritis that has been widely used for preclinical testing ofnumerous anti-arthritic agents (see Trentham, et al., J. Exp. Med.146(3):857-868 (1977), Bendele, et al., Toxicologic Pathol. 27:134-142(1999), Bendele, et al., Arthritis Rheum. 42:498-506 (1999)). Thehallmarks of this model are reliable onset and progression of robust,easily measurable polyarticular inflammation, marked cartilagedestruction in association with pannus formation and mild to moderatebone resorption and periosteal bone proliferation.

Female Lewis rats (approximately 0.2 kilograms) are anesthetized withisoflurane and injected with Freund's Incomplete Adjuvant containing 2mg/mL bovine type II collagen at the base of the tail and two sites onthe back on days 0 and 6 of this 17 day study. A candidate compound isdosed daily in a sub-cutaneous manner from day 0 till day 17 at aefficacious dose. Caliper measurements of the ankle joint diameter weretaken, and reducing joint swelling is taken as a measure of efficacy.

4. Rat Model of Sepsis

To study the effect of compounds of interest on inhibiting thegeneralized inflammatory response that is associated with a sepsis likedisease, the Cecal Ligation and Puncture (CLP) rat model of sepsis isused. A Rat CLP study is conducted essentially as described in FujimuraN, et al. (American Journal Respiratory Critical Care Medicine 2000;161: 440-446). Briefly described here, Wistar Albino Rats of both sexesweighing between 200-250 g are fasted for twelve hours prior toexperiments. Animals are kept on normal 12 hour light and dark cyclesand fed standard rat chow up until 12 hours prior to experiment. Thenanimals are split into four groups; (i) two sham operation groups and(ii) two CLP groups. Each of these two groups (i.e., (i) and (ii)) issplit into vehicle control group and test compound group. Sepsis isinduced by the CLP method. Under brief anesthesia a midline laparotomyis made using minimal dissection and the cecum is ligated just below theileocaecal valve with 3-0 silk, so the intestinal continuity ismaintained. The antimesinteric surface of the cecum is perforated withan 18 gauge needle at two locations 1 cm apart and the cecum is gentlysqueezed until fecal matter is extruded. The bowel is then returned tothe abdomen and the incision is closed. At the end of the operation, allrats are resuscitated with saline, 3 ml/100 g body weight, givensubcutaneously. Postoperatively, the rats are deprived of food, but havefree access to water for the next 16 hours until they are sacrificed.The sham operated groups are given a laparotomy and the cecum ismanipulated but not ligated or perforated. Beneficial effects oftreatment are measured by histopathological scoring of tissues andorgans as well as measurement of several key indicators of hepaticfunction, renal function, and lipid peroxidation. To test for hepaticfunction aspartate transaminase (AST) and alanine transaminase (ALT) aremeasured. Blood urea nitrogen and creatinine concentrations are studiedto assess renal function. Pro-inflammatory cytokines such as TNF-alphaand IL-1beta are also assayed by ELISA for serum levels.

5. Mouse SLE Model of Experimental Lupus Nephritis.

To study the effect of compounds of interest on a Systemic LupusErythematosus (SLE), the MRL/lpr murine SLE model is used. TheMRL/Mp-Tmfrsf6^(lpr/lpr) strain (MRL/lpr) is a commonly used mouse modelof human SLE. To test compounds efficacy in this model male MRL/lpr miceare equally divided between control and C5aR antagonists groups at 13weeks of age. Then over the next 6 weeks compound or vehicle isadministered to the animals via osmotic pumps to maintain coverage andminimize stress effects on the animals. Serum and urine samples arecollected bi-weekly during the six weeks of disease onset andprogression. In a minority of these mice glomerulosclerosis developsleading to the death of the animal from renal failure. Followingmortality as an indicator of renal failure is one of the measuredcriteria and successful treatment will usually result in a delay in theonset of sudden death among the test groups. In addition, the presenceand magnitude of renal disease may also be monitored continuously withblood urea nitrogen (BUN) and albuminuria measurements. Tissues andorgans were also harvested at 19 weeks and subjected to histopathologyand immunohistochemistry and scored based on tissue damage and cellularinfiltration.

6. Rat Model of COPD

Smoke induced airway inflammation in rodent models may be used to assessefficacy of compounds in Chronic Obstructive Pulmonary Disease (COPD).Selective antagonists of chemokines have shown efficacy in this model(see, Stevenson, et al., Am. J. Physiol Lung Cell Mol Physiol. 288L514-L522, (2005)). An acute rat model of COPD is conducted as describedby Stevenson et al. A compound of interest is administered eithersystemically via oral or IV dosing; or locally with nebulized compound.Male Sprague-Dawley rats (350-400 g) are placed in Perspex chambers andexposed to cigarette smoke drawn in via a pump (50 mL every 30 secondswith fresh air in between). Rats are exposed for a total period of 32minutes. Rats are sacrificed up to 7 days after initial exposure. Anybeneficial effects of treatment are assessed by a decrease inflammatorycell infiltrate, decreases in chemokine and cytokine levels.

In a chronic model, mice or rats are exposed to daily tobacco smokeexposures for up to 12 months. Compound is administered systemically viaonce daily oral dosing, or potentially locally via nebulized compound.In addition to the inflammation observed with the acute model (Stevensenet al.), animals may also exhibit other pathologies similar to that seenin human COPD such as emphysema (as indicated by increased mean linearintercept) as well as altered lung chemistry (see Martorana et al, Am.J. Respir. Crit Care Med. 172(7): 848-53.

7. Mouse EAE Model of Multiple Sclerosis

Experimental autoimmune encephalomyelitis (EAE) is a model of humanmultiple sclerosis. Variations of the model have been published, and arewell known in the field. In a typical protocol, C57BL/6 (Charles RiverLaboratories) mice are used for the EAE model. Mice are immunized with200 ug myelin oligodendrocyte glycoprotein (MOG) 35-55 (PeptideInternational) emulsified in Complete Freund's Adjuvant (CFA) containing4 mg/ml Mycobacterium tuberculosis (Sigma-Aldrich) s.c. on day 0. Inaddition, on day 0 and day 2 animals are given 200 ng of pertussis toxin(Calbiochem) i.v. Clinical scoring is based on a scale of 0-5: 0, nosigns of disease; 1, flaccid tail; 2, hind limb weakness; 3, hind limbparalysis; 4, forelimb weakness or paralysis; 5, moribund. Dosing of thecompounds of interest to be assessed can be initiated on day 0(prophylactic) or day 7 (therapeutic, when histological evidence ofdisease is present but few animals are presenting clinical signs) anddosed once or more per day at concentrations appropriate for theiractivity and pharmacokinetic properties, e.g. 100 mg/kg s.c. Efficacy ofcompounds can be assessed by comparisons of severity (maximum meanclinical score in presence of compound compared to vehicle), or bymeasuring a decrease in the number of macrophages (F4/80 positive)isolated from spinal cords. Spinal cord mononuclear cells can beisolated via discontinuous Percoll-gradient. Cells can be stained usingrat anti-mouse F4/80-PE or rat IgG2b-PE (Caltag Laboratories) andquantitated by FACS analysis using 10 ul of Polybeads per sample(Polysciences).

8. Mouse Model of Kidney Transplantation

Transplantation models can be performed in mice, for instance a model ofallogenic kidney transplant from C57BL/6 to BALB/c mice is described inFaikah Gueler et al, JASN Express, Aug. 27, 2008. Briefly, mice areanesthetized and the left donor kidney attached to a cuff of the aortaand the renal vein with a small caval cuff, and the ureters removed enblock. After left nephrectomy of the recipient, the vascular cuffs areanastomosed to the recipient abdominal aorta and vena cava,respectively, below the level of the native renal vessels. The ureter isdirectly anastomosed into the bladder. Cold ischemia time is 60 min, andwarm ischemia time is 30 min. The right native kidney can be removed atthe time of allograft transplantation or at posttransplantation day 4for long-term survival studies. General physical condition of the miceis monitored for evidence of rejection. Compound treatment of animalscan be started before surgery or immediately after transplantation, egby sub cut injection once daily. Mice are studied for renal function andsurvival. Serum creatinine levels are measured by an automated method(Beckman Analyzer, Krefeld, Germany).

9. Mouse Model of Ischemia/Reperfusion

A mouse model of ischemia/reperfusion injury can be performed asdescribed by Xiufen Zheng et al, Am. J. Pathol, Vol 173:4, October,2008. Briefly, CD1 mice aged 6-8 weeks are anesthetized and placed on aheating pad to maintain warmth during surgery. Following abdominalincisions, renal pedicles are bluntly dissected and a microvascularclamp placed on the left renal pedicle for 25-30 minutes. Followingischemia the clamps are removed along with the right kidney, incisionssutured, and the animals allowed to recover. Blood is collected forserum creatinine and BUN analysis as an indicator of kidney health.Alternatively animal survival is monitored over time. Compound can beadministered to animals before and/or after the surgery and the effectson serum creatinine, BUN or animal survival used as indicators ofcompound efficacy.

10. Mouse Model of Tumor Growth

C57BL/6 mice 6-16 weeks of age are injected subcutaneously with 1×105TC-1 cells (ATCC, VA) in the right or left rear flank. Beginning about 2weeks after cell injection, tumors are measured with calipers every 2-4d until the tumor size required the mice are killed. At the time ofsacrifice animals are subjected to a full necropsy and spleens andtumors removed. Excised tumors are measured and weighed. Compounds maybe administered before and/or after tumor injections, and a delay orinhibition of tumor growth used to assess compound efficacy.

In Table 3, below, structures and activity are provided forrepresentative compounds described herein. Activity is provided asfollows for inhibition in the chemotaxis assay (see Example 14 B.3) asdescribed herein: +, 500 nM≤IC₅₀; ++, 50 nM≤IC₅₀<500 nM; +++, 5nM≤IC₅₀<50 nM; and ++++, IC₅₀<5 nM.

TABLE 3 Structure & Biological Activity of Specific Embodiments IC50Compound Structure (nM) 1.001

+++ 1.002

++++ 1.003

+ 1.004

++++ 1.005

+ 1.006

+ 1.007

++ 1.008

++ 1.009

++ 1.010

+++ 1.011

+++ 1.012

+++ 1.013

+++ 1.014

++ 1.015

+++ 1.016

++ 1.017

++++ 1.018

+++ 1.019

+++ 1.020

+++ 1.021

++++ 1.022

+++ 1.023

++++ 1.024

++++ 1.025

++++ 1.026

++++ 1.027

+++ 1.028

++ 1.029

+++ 1.030

+++ 1.031

+++ 1.032

++++ 1.033

+++ 1.034

+++ 1.035

+++ 1.036

+++ 1.037

++++ 1.038

+++ 1.039

+++ 1.040

+ 1.041

++ 1.042

+++ 1.043

+++ 1.044

+++ 1.045

+++ 1.046

+++ 1.047

+++ 1.048

+++ 1.049

++ 1.050

+++ 1.051

+ 1.052

+++ 1.053

++ 1.054

+ 1.055

+ 1.056

+ 1.057

+ 1.058

+ 1.059

+ 1.060

++ 1.061

+ 1.062

+ 1.063

+ 1.064

+ 1.065

+ 1.066

+ 1.067

++

While particular embodiments of this invention are described herein,upon reading the description, variations of the disclosed embodimentsmay become apparent to individuals working in the art, and it isexpected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A compound of Formula (I)

or a pharmaceutically acceptable salt thereof, wherein, ring vertex A¹is selected from the group consisting of N, CH, C(O) and C(R⁴); ringvertex A² is selected from the group consisting of N, CH, and C(R⁴);each of ring vertices A³, A⁴, A⁵ and A⁶ is independently selected fromthe group consisting of CH and C(R⁴); each of the dashed bondsindependently indicate a single or double bond; R¹ is selected from thegroup consisting of —C₁₋₈ alkylene-heteroaryl, —C₁₋₈ alkylene-C₆₋₁₀aryl, C₁₋₈ alkyl, C₁₋₈ haloalkyl, —C(O)—C₁₋₈ alkyl, —C(O)—C₆₋₁₀ aryl,—C(O)-heteroaryl, —C(O)—C₃₋₆ cycloalkyl, —C(O)-heterocycloalkyl,—C(O)NR^(1a)R^(1b), —SO₂—C₆₋₁₀ aryl, —SO₂-heteroaryl, —C(O)—C₁₋₈alkylene-O-heteroaryl, —C(O)—C₁₋₈ alkylene-O—C₆₋₁₀ aryl, —C(O)—C₁₋₈alkylene-O-heterocycloalkyl, —C(O)—C₁₋₈ alkylene-O—C₃₋₆ cycloalkyl,—C(O)—C₁₋₈ alkylene-heteroaryl, —C(O)—C₁₋₈ alkylene-C₆₋₁₀ aryl,—C(O)—C₁₋₈ alkylene-heterocycloalkyl, —C(O)—C₁₋₈ alkylene-C₃₋₆cycloalkyl and —CO₂R^(1a); the heterocycloalkyl is a 4 to 8 memberedring having from 1 to 3 heteroatoms as ring vertices selected from N, Oand S; and the heteroaryl group is a 5 to 10 membered aromatic ringhaving from 1 to 3 heteroatoms as ring vertices selected from N, O andS; wherein R^(1a) and R^(1b) are each independently selected from thegroup consisting of hydrogen, C₁₋₈ alkyl, and C₁₋₈ haloalkyl; wherein R¹is optionally substituted with 1 to 5 R⁵ substituents; R^(2a) and R^(2e)are each independently selected from the group consisting of C₁₋₆ alkyl,C₁₋₆alkoxy, C₁₋₆ haloalkyl, —O—C₁₋₆ haloalkyl, —S—C₁₋₆ alkyl, —C₁₋₆alkyl-O—C₁₋₆ alkyl, —C₁₋₆ alkyl-S—C₁₋₆ alkyl, CN, and halogen; R^(2b),R^(2c), and R^(2d) are each independently selected from the groupconsisting of hydrogen, C₁₋₆ alkyl, C₁₋₆ alkoxy, C₁₋₆ haloalkyl, —O—C₁₋₆haloalkyl, —S—C₁₋₆ alkyl, —C₁₋₆ alkyl-O—C₁₋₆ alkyl, —C₁₋₆ alkyl-S—C₁₋₆alkyl, cyano, and halogen; each R³ is independently selected from thegroup consisting of C₁₋₆ alkyl, C₁₋₆ haloalkyl, halogen and hydroxyl,and optionally two R³ groups on the same carbon atom are combined toform oxo (═O) or to form a three to five membered cycloalkyl ring; eachR⁴ is independently selected from the group consisting of C₁₋₆ alkyl,C₁₋₆ alkoxy, C₁₋₆ hydroxyalkyl, C₁₋₆ haloalkyl, C₁₋₆ haloalkoxy, —O—C₁₋₆haloalkyl, halogen, cyano, hydroxyl, —S—C₁₋₆ alkyl, —C₁₋₆ alkyl-O—C₁₋₆alkyl, —C₁₋₆ alkyl-S—C₁₋₆ alkyl, —NR^(4a)R^(4b), —CONR^(4a)R^(4b),—CO₂R^(4a), —COR^(4a), —OC(O)NR^(4a)R^(4b), —NR^(4a)C(O)R^(4b),—NR^(4a)C(O)₂R^(4b), and —NR^(4a)—C(O)NR^(4a)R^(4b); each R^(4a) andR^(4b) is independently selected from the group consisting of hydrogen,C₁₋₄ alkyl, and C₁₋₄ haloalkyl; each R⁵ is independently selected fromthe group consisting of C₁₋₈ alkyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₁₋₈haloalkoxy, C₁₋₈ hydroxyalkyl, —C₁₋₈ alkylene-heterocycloalkyl, —C₁₋₈alkylene-C₃₆ cycloalkyl, C₃₋₆ cycloalkyl, heterocycloalkyl, halogen, OH,C₂₋₈ alkenyl, C₂₋₈ alkynyl, CN, C(O)R^(5a), —NR^(5b)C(O)R^(5a),—CONR^(5a)R^(5b), —NR^(5a)R^(5b), —C₁₋₈ alkylene-NR^(5a)R^(5b), —S—C₁₋₆alkyl, —C₁₋₆ alkylene-O—C₁₋₆ alkyl, —C₁₋₆ alkylene-S—C₁₋₆ alkyl,—OC(O)NR^(5a)R^(5b), —NR^(5a)C(O)₂R^(5b), —NR^(5a)—C(O)NR^(5b)R^(5b),and CO₂R^(5a); wherein the heterocycloalkyl group is a 4 to 8 memberedring having from 1 to 3 heteroatoms as ring vertices selected from N, O,and S; wherein each R^(5a) and R^(5b) is independently selected from thegroup consisting of hydrogen, C₁₋₈ alkyl, and C₁₋₈ haloalkyl, or whenR^(5a) and R^(5b) are attached to the same nitrogen atom they arecombined with the nitrogen atom to form a 5 or 6-membered ring havingfrom 0 to 1 additional heteroatoms as ring vertices selected from N, O,or S; and the subscript n is 0, 1, 2 or
 3. 2. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein the ring portionhaving A¹, A², A³, A⁴, A⁵, and A⁶ as ring vertices is a bicyclicheteroaryl selected from the group consisting of

wherein m is 0, 1, 2 or 3; and wherein the R⁴ substituents may beattached to any suitable carbon ring vertex of the bicyclic heteroaryl.3. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶ asring vertices is a bicyclic heteroaryl represented by the structure

wherein m is 0, 1, 2, or 3; and wherein the R⁴ substituents may beattached to any suitable carbon ring vertex of the bicyclic heteroaryl.4. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein each R⁴ is independently selected from the groupconsisting of C₁₋₄ alkyl, C₁₋₄ alkoxy, C₁₋₆ hydroxyalkyl, halogen,cyano, and —CO₂R^(4a); and wherein the R⁴ substituents may be attachedto any suitable carbon ring vertex of the bicyclic heteroaryl.
 5. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶ as ringvertices is a bicyclic heteroaryl selected from the group consisting of:


6. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein the ring portion having A¹, A², A³, A⁴, A⁵, and A⁶ asring vertices is a bicyclic heteroaryl selected from the groupconsisting of:


7. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is selected from the group consisting of —C₁₋₈alkylene-heteroaryl, —C₁₋₈alkylene-C₆₋₁₀ aryl, C₁₋₈ alkyl, C₁₋₈haloalkyl, —C(O)—C₁₋₈ alkyl, —C(O)—C₆₋₁₀ aryl, —C(O)-heteroaryl,—C(O)—C₃₋₈ cycloalkyl, —C(O)NR^(1a)R^(1b), —SO₂—C₆₋₁₀ aryl, —C(O)—C₁₋₈alkylene-O—C₆₋₁₀ aryl, and —CO₂R^(1a); wherein the heterocycloalkyl is a4 to 8 membered ring having from 1 to 3 heteroatoms as ring verticesselected from N, O and S; and the heteroaryl group is a 5 to 10 memberedaromatic ring having from 1 to 3 heteroatoms as ring vertices selectedfrom N, O and S; and wherein R^(1a) and R^(1b) are each independentlyselected from the group consisting of hydrogen, C₁₋₈ alkyl, and C_(1-s)haloalkyl; and R¹ is optionally substituted with 1 to 5 R⁵ substituents.8. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein each R⁵ is independently selected from the groupconsisting of C₁₋₈ alkyl, C₁₋₈ alkoxy, C₁₋₈ haloalkyl, C₁₋₈ haloalkoxy,C₁₋₈ hydroxyalkyl, C₃₋₆ cycloalkyl, halogen, OH, —NR^(5a)R^(5b), andCO₂R^(5a), wherein each R^(5a) and R^(5b) is independently selected fromthe group consisting of hydrogen, C₁₋₈ alkyl, and C₁₋₈ haloalkyl.
 9. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R¹ is —CH₂-phenyl optionally substituted by 1 to 3 R⁵.
 10. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein R¹ is —CH₂-phenyl substituted by 1 or 2 R⁵, wherein each R⁵ isindependently C₁₋₄ haloalkyl.
 11. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is —CH₂-phenylsubstituted by 1 or 2 CF₃.
 12. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein R¹ is selected fromthe group consisting of


13. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R¹ is


14. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein R^(2b), R^(2c), and R^(2d) are each H.
 15. The compoundof claim 1, or a pharmaceutically acceptable salt thereof, whereinR^(2a) and R^(2e) are each independently selected from the groupconsisting of C₁₋₆ alkyl and C₁₋₆ haloalkyl.
 16. The compound of claim1, or a pharmaceutically acceptable salt thereof, wherein R^(2a) andR^(2e) are each independently C₁₋₆ alkyl.
 17. The compound of claim 1,or a pharmaceutically acceptable salt thereof, wherein R^(2a) and R^(2e)are each independently selected from the group consisting of methyl andethyl.
 18. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein R^(2a) and R^(2e) are both methyl or are bothethyl.
 19. The compound of claim 1, or a pharmaceutically acceptablesalt thereof, wherein

is selected from the group consisting of


20. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein n is 0, 1 or
 2. 21. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein each R³ isindependently C₁₋₄ alkyl.
 22. The compound of claim 1, or apharmaceutically acceptable salt thereof, wherein

is selected from the group consisting of


23. The compound of claim 1, or a pharmaceutically acceptable saltthereof, having a structure represented by Formula (Ia), (Ib) or (Ic):


24. The compound of claim 2, or a pharmaceutically acceptable saltthereof, having a structure represented by Formula (Id), (Ie), or (If):

wherein m is 0, 1 or 2; wherein the R⁴ substituents may be attached toany suitable carbon ring vertex of the bicyclic heteroaryl; and whereinp is 0, 1 or
 2. 25. The compound of claim 2, or a pharmaceuticallyacceptable salt thereof, having a structure represented by Formula (Ig)or (Ih):

wherein m is 0, 1 or 2 and wherein the R⁴ substituents may be attachedto any suitable carbon ring vertex of the indole heteroaryl.
 26. Thecompound of claim 1, or a pharmaceutically acceptable salt thereof,wherein said compound is selected from the group consisting of


27. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein said compound is selected from the group consisting of


28. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein said compound is selected from the group consisting of


29. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein said compound is


30. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein said compound is


31. The compound of claim 1, or a pharmaceutically acceptable saltthereof, wherein said compound is